Fernando Almazán - Academia.edu (original) (raw)
Papers by Fernando Almazán
Analytical Chemistry, Jul 18, 2023
Signal Transduction and Targeted Therapy, Jul 3, 2023
Dear Editor, One of the most serious issues in modern oncology is the ineffectiveness of treatmen... more Dear Editor, One of the most serious issues in modern oncology is the ineffectiveness of treatments in destroying tumours, which leads to tumour recurrence and, ultimately, patient death. This phenomenon is caused by conventional therapies' fractional killing of tumour cells, which can also cause resistant cells to spread. 1 We analysed chemotherapy-resistant cells and discovered that they are smaller than untreated cells (Fig. 1a). The fact that this phenomenon occurs in all tested cell types (Hela, A549, Huh7, and MCF7) and regardless of the chemotherapy regimen (TRAIL, Camptothecin, Doxorubicin) suggests that it is a widespread phenomenon (Fig. 1a, Supplementary Fig. S1). Except for Doxorubicin, which causes cells to accumulate in G1 phase of the cell cycle, this reduction in the cell volume of resistant cells is not caused by selective cell death at a specific stage of the cell cycle (Fig. 1b). Nor is it due to the selection of small cells, as with all treatments we find a population of small cells that does not exist in untreated cells, gray area (Fig. 1a). The alteration in the biosynthesis and/or degradation of proteins, which are the macromolecules that contribute most to cell size, may therefore be the cause of the shrinkage. We then analyzed protein synthesis using puromycin, which is incorporated into nascent proteins, and UbiQ-018, which is a fluorescent reagent used to measure proteasomal activity. Hela cells resistant to TRAIL treatment (24 h, 30 ng/ml) have a higher concentration of protein synthesis activity, as well as higher proteasomal activity than untreated cells (Fig. 1c). We measured proteostatic activity (protein synthesis and degradation) 4 h after starting the treatments, when cell death had not yet occurred. The treatments used in this study increased protein synthesis (Fig. 1d). For degradation studies we focused on the effect of camptothecin which, like translation, increases activity (Supplementary Fig. S2). Changes in the protein synthesis/degradation balance caused by camptothecin correlate with the observed cell volume variation (Fig. 1e), supporting the hypothesis that cell size changes are caused by a change in the proteostatic balance. Protein synthesis may be regulated at multiple levels. The mTOR pathway impacts translation. Phosphorylation of ribosomal protein S6 at Ser240 (PS6) is a target of mTOR and modulates protein synthesis. Treatments increased PS6 levels that correlate with changes in puromycin incorporation (Fig. 1f). Furthermore, when we inhibited mTOR with rapamycin (1 μM) exposure to TRAIL 30 ng did not induce changes in translational activity. These results altogether suggest that the increase in protein synthesis after treatments is a consequence of mTOR activation (Fig. 1g). Next, we investigate whether high proteostatic activity (protein synthesis and degradation) is required for surviving apoptotic signals. When cells were treated with TRAIL or camptothecin as well as translation inhibitors, a strong synergistic effect was
bioRxiv (Cold Spring Harbor Laboratory), Jul 22, 2020
An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in Decembe... more An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.
Journal of Virology, May 1, 1990
A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ,... more A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ, and RD' of African swine fever virus DNA were mapped and sequenced. Analysis of these sequences revealed the presence of a family of homologous open reading frames in regions close to the DNA ends. The whole family is composed of six open reading frames with an average length of 360 coding triplets (multigene family 360), four of which are located in the left part of the genome and two of which are in the right terminal EcoRI fragment. In dose proximity to the right terminal inverted repeat, we found an additional small open reading frame which was homologous to the 5'-terminal portion of the other open reading frames, suggesting that most of that open reading frame has been deleted. These repeated sequences account for the previously described inverted internal repetitions (J. M. Sogo, J. M. Almendral, A. Talavera, and E. Vinuela, Virology 133:271-275, 1984). Most of the genes of multigene family 360 are transcribed in African swine fever virus-infected cells. A comparison of the predicted protein sequences of family 360 indicated that several residues are conserved, suggesting that an overall structure is maintained for every member of the family. The transcription direction of each open reading frame, as well as the evolutionary relationships among the genes, suggests that the family originated by gene duplication and translocation of sequences between the DNA ends.
Advances in Experimental Medicine and Biology, 2006
Aerosol Science and Technology
The conical inner electrode of a recently described differential mobility analyzer (DMA), coverin... more The conical inner electrode of a recently described differential mobility analyzer (DMA), covering at resolution >50 the size range up to 60 nm, has been modified by increasing its radius at the outlet slit from 1 cm to 1.6 cm, and decreasing its convergence angle from 3 to 0.5. The cylindrical outer electrode radius is maintained at 2 cm, while other critical dimensions are only slightly modified. The classification voltage for fixed flow rate of sheath gas and particle diameter decreases almost fourfold, extending the size range theoretically spanned at high resolution well beyond 120 nm. Tests with multiply charged polystyrene latex spheres 200 nm in diameter confirm the actual ability to cover this wide size range. A resolution above 100 (with no corrections for instrument or particle nonidealities) is demonstrated with rotavirus double-layered particles (DLPs), whose mobility diameter is found to be near 60 nm. The DLP has several configurations that complicate its use for DMA characterization, but occasionally presents mainly one conformer, with singularly narrow relative mobility width FWHM < 1/150. Two broad range instruments with slightly different axial lengths have been developed and tested, both reaching resolving powers of 100.
Immunology, 1999
Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on... more Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I‐mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane‐bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane‐bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs w...
Virus Research, Dec 1, 2014
Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, ... more 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).
Frontiers in Microbiology
Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell... more Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3′-untranslated region (3’-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3’UTR of SERINC5 mRNA...
Viruses, 2020
Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently... more 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 infect...
Scientific Reports, 2019
Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to ... more 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 ...
La Ciencia Y Tecnologia Ante El Tercer Milenio Vol 1 2002 Isbn 84 95486 37 7 Pags 465 482, 2002
Advances in Experimental Medicine and Biology, 2001
Veterinary Research, 2013
Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (P... more 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 predomi...
Journal of Virology, 2005
During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone,... more During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein...
Journal of Biotechnology, 2008
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Fecha de presantación internacional: 23.06.2006.- Titular: Consejo Superior de Investigaciones Ci... more Fecha de presantación internacional: 23.06.2006.- Titular: Consejo Superior de Investigaciones Científicas (CSIC)The present invention relates to nucleic acids encoding attenuated SARS-CoV viruses which are capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 2 when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture. According to a further aspect of the present invention, the nucleic acids encoding an attenuated SARS-CoV virus, are obtainable by a method comprising steps, wherein the genome of a SARS-CoV virus is modified by amending the sequence of the gene encoding the SARS-CoV E protein so that the nucleic acid cannot express a functional E protein. The present invention further relates to the viruses encoded by these nucleic acids as well as the medical use of the nucleic acids and of the viruses.Peer reviewe
Journal of Visualized Experiments
The association of Zika virus (ZIKV) infection with neurological complications during the recent ... more The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation.
Veterinary Research, 2017
After publication of the article [1], it has been brought to our attention that an acknowledgemen... more 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. "
Analytical Chemistry, Jul 18, 2023
Signal Transduction and Targeted Therapy, Jul 3, 2023
Dear Editor, One of the most serious issues in modern oncology is the ineffectiveness of treatmen... more Dear Editor, One of the most serious issues in modern oncology is the ineffectiveness of treatments in destroying tumours, which leads to tumour recurrence and, ultimately, patient death. This phenomenon is caused by conventional therapies' fractional killing of tumour cells, which can also cause resistant cells to spread. 1 We analysed chemotherapy-resistant cells and discovered that they are smaller than untreated cells (Fig. 1a). The fact that this phenomenon occurs in all tested cell types (Hela, A549, Huh7, and MCF7) and regardless of the chemotherapy regimen (TRAIL, Camptothecin, Doxorubicin) suggests that it is a widespread phenomenon (Fig. 1a, Supplementary Fig. S1). Except for Doxorubicin, which causes cells to accumulate in G1 phase of the cell cycle, this reduction in the cell volume of resistant cells is not caused by selective cell death at a specific stage of the cell cycle (Fig. 1b). Nor is it due to the selection of small cells, as with all treatments we find a population of small cells that does not exist in untreated cells, gray area (Fig. 1a). The alteration in the biosynthesis and/or degradation of proteins, which are the macromolecules that contribute most to cell size, may therefore be the cause of the shrinkage. We then analyzed protein synthesis using puromycin, which is incorporated into nascent proteins, and UbiQ-018, which is a fluorescent reagent used to measure proteasomal activity. Hela cells resistant to TRAIL treatment (24 h, 30 ng/ml) have a higher concentration of protein synthesis activity, as well as higher proteasomal activity than untreated cells (Fig. 1c). We measured proteostatic activity (protein synthesis and degradation) 4 h after starting the treatments, when cell death had not yet occurred. The treatments used in this study increased protein synthesis (Fig. 1d). For degradation studies we focused on the effect of camptothecin which, like translation, increases activity (Supplementary Fig. S2). Changes in the protein synthesis/degradation balance caused by camptothecin correlate with the observed cell volume variation (Fig. 1e), supporting the hypothesis that cell size changes are caused by a change in the proteostatic balance. Protein synthesis may be regulated at multiple levels. The mTOR pathway impacts translation. Phosphorylation of ribosomal protein S6 at Ser240 (PS6) is a target of mTOR and modulates protein synthesis. Treatments increased PS6 levels that correlate with changes in puromycin incorporation (Fig. 1f). Furthermore, when we inhibited mTOR with rapamycin (1 μM) exposure to TRAIL 30 ng did not induce changes in translational activity. These results altogether suggest that the increase in protein synthesis after treatments is a consequence of mTOR activation (Fig. 1g). Next, we investigate whether high proteostatic activity (protein synthesis and degradation) is required for surviving apoptotic signals. When cells were treated with TRAIL or camptothecin as well as translation inhibitors, a strong synergistic effect was
bioRxiv (Cold Spring Harbor Laboratory), Jul 22, 2020
An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in Decembe... more An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.
Journal of Virology, May 1, 1990
A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ,... more A group of cross-hybridizing DNA segments contained within the restriction fragments RK', RL, RJ, and RD' of African swine fever virus DNA were mapped and sequenced. Analysis of these sequences revealed the presence of a family of homologous open reading frames in regions close to the DNA ends. The whole family is composed of six open reading frames with an average length of 360 coding triplets (multigene family 360), four of which are located in the left part of the genome and two of which are in the right terminal EcoRI fragment. In dose proximity to the right terminal inverted repeat, we found an additional small open reading frame which was homologous to the 5'-terminal portion of the other open reading frames, suggesting that most of that open reading frame has been deleted. These repeated sequences account for the previously described inverted internal repetitions (J. M. Sogo, J. M. Almendral, A. Talavera, and E. Vinuela, Virology 133:271-275, 1984). Most of the genes of multigene family 360 are transcribed in African swine fever virus-infected cells. A comparison of the predicted protein sequences of family 360 indicated that several residues are conserved, suggesting that an overall structure is maintained for every member of the family. The transcription direction of each open reading frame, as well as the evolutionary relationships among the genes, suggests that the family originated by gene duplication and translocation of sequences between the DNA ends.
Advances in Experimental Medicine and Biology, 2006
Aerosol Science and Technology
The conical inner electrode of a recently described differential mobility analyzer (DMA), coverin... more The conical inner electrode of a recently described differential mobility analyzer (DMA), covering at resolution >50 the size range up to 60 nm, has been modified by increasing its radius at the outlet slit from 1 cm to 1.6 cm, and decreasing its convergence angle from 3 to 0.5. The cylindrical outer electrode radius is maintained at 2 cm, while other critical dimensions are only slightly modified. The classification voltage for fixed flow rate of sheath gas and particle diameter decreases almost fourfold, extending the size range theoretically spanned at high resolution well beyond 120 nm. Tests with multiply charged polystyrene latex spheres 200 nm in diameter confirm the actual ability to cover this wide size range. A resolution above 100 (with no corrections for instrument or particle nonidealities) is demonstrated with rotavirus double-layered particles (DLPs), whose mobility diameter is found to be near 60 nm. The DLP has several configurations that complicate its use for DMA characterization, but occasionally presents mainly one conformer, with singularly narrow relative mobility width FWHM < 1/150. Two broad range instruments with slightly different axial lengths have been developed and tested, both reaching resolving powers of 100.
Immunology, 1999
Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on... more Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I‐mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane‐bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane‐bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs w...
Virus Research, Dec 1, 2014
Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, ... more 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).
Frontiers in Microbiology
Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell... more Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3′-untranslated region (3’-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3’UTR of SERINC5 mRNA...
Viruses, 2020
Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently... more 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 infect...
Scientific Reports, 2019
Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to ... more 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 ...
La Ciencia Y Tecnologia Ante El Tercer Milenio Vol 1 2002 Isbn 84 95486 37 7 Pags 465 482, 2002
Advances in Experimental Medicine and Biology, 2001
Veterinary Research, 2013
Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (P... more 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 predomi...
Journal of Virology, 2005
During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone,... more During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein...
Journal of Biotechnology, 2008
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Fecha de presantación internacional: 23.06.2006.- Titular: Consejo Superior de Investigaciones Ci... more Fecha de presantación internacional: 23.06.2006.- Titular: Consejo Superior de Investigaciones Científicas (CSIC)The present invention relates to nucleic acids encoding attenuated SARS-CoV viruses which are capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 2 when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture. According to a further aspect of the present invention, the nucleic acids encoding an attenuated SARS-CoV virus, are obtainable by a method comprising steps, wherein the genome of a SARS-CoV virus is modified by amending the sequence of the gene encoding the SARS-CoV E protein so that the nucleic acid cannot express a functional E protein. The present invention further relates to the viruses encoded by these nucleic acids as well as the medical use of the nucleic acids and of the viruses.Peer reviewe
Journal of Visualized Experiments
The association of Zika virus (ZIKV) infection with neurological complications during the recent ... more The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation.
Veterinary Research, 2017
After publication of the article [1], it has been brought to our attention that an acknowledgemen... more 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. "