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1.
Mol Ecol ; 31(5): 1444-1460, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34905257

RESUMEN

In animals with distinct life stages such as holometabolous insects, adult phenotypic variation is often shaped by the environment of immature stages, including their interactions with microbes colonizing larval habitats. Such carry-over effects were previously observed for several adult traits of the mosquito Aedes aegypti after larval exposure to different bacteria, but the mechanistic underpinnings are unknown. Here, we investigated the molecular changes triggered by gnotobiotic larval exposure to different bacteria in Ae. aegypti. We initially screened a panel of 16 bacterial isolates from natural mosquito breeding sites to determine their ability to influence adult life-history traits. We subsequently focused on four bacterial isolates (belonging to Flavobacterium, Lysobacter, Paenibacillus, and Enterobacteriaceae) with significant carry-over effects on adult survival and found that they were associated with distinct transcriptomic profiles throughout mosquito development. Moreover, we detected carry-over effects at the level of gene expression for the Flavobacterium and Paenibacillus isolates. The most prominent transcriptomic changes in gnotobiotic larvae reflected a profound remodelling of lipid metabolism, which translated into phenotypic differences in lipid storage and starvation resistance at the adult stage. Together, our findings indicate that larval exposure to environmental bacteria trigger substantial physiological changes that impact adult fitness, uncovering a possible mechanism underlying carry-over effects of mosquito-bacteria interactions during larval development.


Asunto(s)
Aedes , Aedes/microbiología , Animales , Bacterias/genética , Ecosistema , Larva/microbiología
2.
J Virol ; 93(18)2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31243123

RESUMEN

Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses.IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.


Asunto(s)
Flavivirus/metabolismo , Mosquitos Vectores/virología , Interferencia Viral/fisiología , Aedes/virología , Animales , Arbovirus/metabolismo , Línea Celular , Dengue/virología , Virus del Dengue/aislamiento & purificación , Virus del Dengue/metabolismo , Flavivirus/genética , Flavivirus/aislamiento & purificación , Humanos , Virus de Insectos , Filogenia , Replicación Viral/fisiología , Virus Zika/aislamiento & purificación , Virus Zika/metabolismo , Infección por el Virus Zika/virología
3.
PLoS Pathog ; 11(3): e1004738, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25734338

RESUMEN

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5-10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.


Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Encefalitis Transmitida por Garrapatas/prevención & control , Vacunas Virales/genética , Animales , Modelos Animales de Enfermedad , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos C57BL , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/inmunología
4.
J Neurosci ; 35(6): 2817-29, 2015 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-25673868

RESUMEN

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimer's disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntington's disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.


Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Astrocitos , Enfermedad de Huntington/fisiopatología , Quinasas Janus , Factor de Transcripción STAT3 , Transducción de Señal , Enfermedad de Alzheimer/patología , Animales , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Enfermedad de Huntington/patología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética
5.
PLoS Pathog ; 9(2): e1003172, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23436995

RESUMEN

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.


Asunto(s)
Virus Chikungunya/genética , Codón/genética , Aptitud Genética , Genoma Viral/genética , Replicación Viral , Aedes/virología , Infecciones por Alphavirus/transmisión , Infecciones por Alphavirus/virología , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Fiebre Chikungunya , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/fisiología , Chlorocebus aethiops , Secuencia de Consenso , Evolución Molecular , Variación Genética , Humanos , Mutación , Primates/virología , ARN Viral/biosíntesis , ARN Viral/genética , Análisis de Secuencia de ADN , Pase Seriado , Células Vero , Proteínas Virales/biosíntesis , Proteínas Virales/genética
6.
J Gen Virol ; 95(Pt 11): 2462-2467, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25053561

RESUMEN

Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.


Asunto(s)
Virus ARN/genética , Virus ARN/fisiología , Genética Inversa/métodos , Animales , Línea Celular , Cricetinae , ADN Complementario/genética , ADN Viral/genética , Flavivirus/genética , Flavivirus/fisiología , Genoma Viral , Humanos , Datos de Secuencia Molecular , ARN Viral/genética , Replicación Viral/genética , Replicación Viral/fisiología
7.
Emerg Microbes Infect ; 10(1): 1346-1357, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34139961

RESUMEN

Dengue virus (DENV) evolutionary dynamics are characterized by frequent DENV genotype/lineage replacements, potentially associated with changes in disease severity and human immunity. New Caledonia (NC) and Cambodia, two contrasted epidemiological settings, respectively experienced a DENV-1 genotype IV to I replacement in 2012 and a DENV-1 genotype I lineage 3-4 replacement in 2005-2007, both followed by a massive dengue outbreak. However, their underlying evolutionary drivers have not been elucidated. Here, we tested the hypothesis that these genotype/lineage switches reflected a higher transmission fitness of the replacing DENV genotype/lineage in the mosquito vector using in vivo competition experiments. For this purpose, field-derived Aedes aegypti from NC and Cambodia were orally challenged with epidemiologically relevant pairs of four DENV-1 genotype I and IV strains from NC or four DENV-1 genotype I lineage 3 and 4 strains from Cambodia, respectively. The relative transmission fitness of each DENV-1 genotype/lineage was measured by quantitative RT-PCR for infection, dissemination, and transmission rates. Results showed a clear transmission fitness advantage of the replacing DENV-1 genotype I from NC within the vector. A similar but more subtle pattern was observed for the DENV-1 lineage 4 replacement in Cambodia. Our results support the hypothesis that vector-driven selection contributed to the DENV-1 genotype/lineage replacements in these two contrasted epidemiological settings, and reinforce the idea that natural selection taking place within the mosquito vector plays an important role in DENV short-term evolutionary dynamics.


Asunto(s)
Aedes/virología , Virus del Dengue/genética , Dengue/virología , Mosquitos Vectores/virología , Selección Genética , Animales , Cambodia/epidemiología , Dengue/epidemiología , Dengue/transmisión , Virus del Dengue/fisiología , Brotes de Enfermedades , Aptitud Genética , Genotipo , Humanos , Nueva Caledonia/epidemiología , Filogenia , Saliva/virología
8.
Nat Commun ; 12(1): 916, 2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33568638

RESUMEN

The global emergence of Zika virus (ZIKV) revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compare seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We find that recent African ZIKV strains display higher transmissibility in mosquitoes and higher lethality in both adult and fetal mice than their Asian counterparts. We emphasize the high epidemic potential of African ZIKV strains and suggest that they could more easily go unnoticed by public health surveillance systems than Asian strains due to their propensity to cause fetal loss rather than birth defects.


Asunto(s)
Infección por el Virus Zika/mortalidad , Infección por el Virus Zika/virología , Virus Zika/fisiología , Virus Zika/patogenicidad , Aedes/fisiología , Aedes/virología , África , Animales , Asia , Femenino , Humanos , Masculino , Ratones , Filogenia , Virulencia , Virus Zika/clasificación , Virus Zika/genética , Infección por el Virus Zika/transmisión
9.
Sci Rep ; 10(1): 7750, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-32385369

RESUMEN

Many emerging arboviruses of global public health importance, such as dengue virus (DENV) and yellow fever virus (YFV), originated in sylvatic transmission cycles involving wild animals and forest-dwelling mosquitoes. Arbovirus emergence in the human population typically results from spillover transmission via bridge vectors, which are competent mosquitoes feeding on both humans and wild animals. Another related, but less studied concern, is the risk of 'spillback' transmission from humans into novel sylvatic cycles. We colonized a sylvatic population of Aedes malayensis from a forested area of the Nakai district in Laos to evaluate its potential as an arbovirus bridge vector. We found that this Ae. malayensis population was overall less competent for DENV and YFV than an urban population of Aedes aegypti. Olfactometer experiments showed that our Ae. malayensis colony did not display any detectable attraction to human scent in laboratory conditions. The relatively modest vector competence for DENV and YFV, combined with a lack of detectable attraction to human odor, indicate a low potential for this sylvatic Ae. malayensis population to act as an arbovirus bridge vector. However, we caution that opportunistic blood feeding on humans by sylvatic Ae. malayensis may occasionally contribute to bridge sylvatic and human transmission cycles.


Asunto(s)
Aedes/fisiología , Arbovirus/fisiología , Mosquitos Vectores/fisiología , Aedes/virología , Animales , Conservación de los Recursos Naturales , Humanos , Laos , Mosquitos Vectores/virología , Odorantes , Riesgo , Especificidad de la Especie
10.
Sci Rep ; 10(1): 18404, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-33110109

RESUMEN

In most of the world, Dengue virus (DENV) is mainly transmitted by the mosquito Aedes aegypti while in Europe, Aedes albopictus is responsible for human DENV cases since 2010. Identifying mutations that make DENV more competent for transmission by Ae. albopictus will help to predict emergence of epidemic strains. Ten serial passages in vivo in Ae. albopictus led to select DENV-1 strains with greater infectivity for this vector in vivo and in cultured mosquito cells. These changes were mediated by multiple adaptive mutations in the virus genome, including a mutation at position 10,418 in the DENV 3'UTR within an RNA stem-loop structure involved in subgenomic flavivirus RNA production. Using reverse genetics, we showed that the 10,418 mutation alone does not confer a detectable increase in transmission efficiency in vivo. These results reveal the complex adaptive landscape of DENV transmission by mosquitoes and emphasize the role of epistasis in shaping evolutionary trajectories of DENV variants.


Asunto(s)
Adaptación Fisiológica , Aedes/virología , Virus del Dengue/fisiología , Mosquitos Vectores/virología , Animales , Dengue/epidemiología , Dengue/transmisión , Epistasis Genética , Humanos
11.
Science ; 370(6519): 991-996, 2020 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-33214283

RESUMEN

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito Aedes aegypti is a major arbovirus vector native to Africa that invaded most of the world's tropical belt over the past four centuries, after the evolution of a "domestic" form that specialized in biting humans and breeding in water storage containers. Here, we show that human specialization and subsequent spread of A. aegypti out of Africa were accompanied by an increase in its intrinsic ability to acquire and transmit the emerging human pathogen Zika virus. Thus, the recent evolution and global expansion of A. aegypti promoted arbovirus emergence not solely through increased vector-host contact but also as a result of enhanced vector susceptibility.


Asunto(s)
Aedes/virología , Interacciones Microbiota-Huesped/genética , Mosquitos Vectores/virología , Infección por el Virus Zika/transmisión , Virus Zika/fisiología , Aedes/genética , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Mosquitos Vectores/genética
12.
PLoS Negl Trop Dis ; 13(10): e0007783, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31589616

RESUMEN

The case-fatality rate of yellow fever virus (YFV) is one of the highest among arthropod-borne viruses (arboviruses). Although historically, the Asia-Pacific region has remained free of YFV, the risk of introduction has never been higher due to the increasing influx of people from endemic regions and the recent outbreaks in Africa and South America. Singapore is a global hub for trade and tourism and therefore at high risk for YFV introduction. Effective control of the main domestic mosquito vector Aedes aegypti in Singapore has failed to prevent re-emergence of dengue, chikungunya and Zika viruses in the last two decades, raising suspicions that peridomestic mosquito species untargeted by domestic vector control measures may contribute to arbovirus transmission. Here, we provide empirical evidence that the peridomestic mosquito Aedes malayensis found in Singapore can transmit YFV. Our laboratory mosquito colony recently derived from wild Ae. malayensis in Singapore was experimentally competent for YFV to a similar level as Ae. aegypti controls. In addition, we captured Ae. malayensis females in one human-baited trap during three days of collection, providing preliminary evidence that host-vector contact may occur in field conditions. Finally, we detected Ae. malayensis eggs in traps deployed in high-rise building areas of Singapore. We conclude that Ae. malayensis is a competent vector of YFV and re-emphasize that vector control methods should be extended to target peridomestic vector species.


Asunto(s)
Aedes/virología , Mosquitos Vectores/virología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/fisiología , Aedes/crecimiento & desarrollo , Animales , Femenino , Interacciones Huésped-Patógeno/fisiología , Humanos , Mosquitos Vectores/fisiología , Saliva/virología , Singapur/epidemiología , Fiebre Amarilla/epidemiología , Fiebre Amarilla/transmisión
13.
Emerg Microbes Infect ; 7(1): 161, 2018 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-30254297

RESUMEN

Zika virus (ZIKV) has recently become dispersed throughout the tropics and sub-tropics, causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. In this study, we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared different constructs and confirmed the need to modify the cleavage site between the pre-peptide and prM protein. Genotypic characterization of the chimeras indicated that the emergence of compensatory mutations in the E protein was required to restore viral replicative fitness. Using an immunocompromised mouse model, we demonstrated that mice infected with the chimeric virus produced levels of neutralizing antibodies that were close to those observed following infection with ZIKV. Furthermore, pre-immunized mice were protected against viscerotropic and neuroinvasive disease following challenge with a heterologous ZIKV strain. These data provide a sound basis for the future development of this ZIKV vaccine candidate.


Asunto(s)
Vacunas Virales/inmunología , Virus de la Fiebre Amarilla/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Cricetinae , Femenino , Humanos , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Virus de la Fiebre Amarilla/genética , Virus Zika/genética , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología
14.
Emerg Microbes Infect ; 7(1): 40, 2018 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-29559627

RESUMEN

Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated 'SuPReMe' (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.


Asunto(s)
Virus Chikungunya/genética , ARN/genética , Genética Inversa/métodos , Línea Celular , Fiebre Chikungunya/virología , Virus Chikungunya/fisiología , Genoma Viral , Humanos , ARN/metabolismo , Replicación Viral
15.
PLoS One ; 13(2): e0193069, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29438402

RESUMEN

Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons) method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR) 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV) and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.


Asunto(s)
Genoma Viral , Infecciones por Virus ARN/genética , Virus ARN/genética , ARN Viral/genética , Genética Inversa/métodos , Animales , Humanos , Virión/genética , Replicación Viral/genética
16.
Sci Rep ; 7(1): 13983, 2017 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-29070887

RESUMEN

Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus). This method represents an effective option to potentially overcome technological issues related to the study of arboviruses.


Asunto(s)
Infecciones por Arbovirus/genética , Infecciones por Arbovirus/virología , Arbovirus/genética , Culicidae/virología , Genética Inversa/métodos , Transfección/métodos , Replicación Viral , Animales , Humanos , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/virología , Virus ARN/genética
17.
PLoS One ; 11(8): e0159564, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27548676

RESUMEN

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.


Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Codón/química , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/prevención & control , Genética Inversa/métodos , Vacunas Virales/genética , Animales , Línea Celular , Codón/metabolismo , Cricetinae , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/mortalidad , Encefalitis Transmitida por Garrapatas/virología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Sistemas de Lectura Abierta , ARN Helicasas/genética , ARN Helicasas/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Análisis de Supervivencia , Vacunas Atenuadas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología
18.
Antiviral Res ; 114: 67-85, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25512228

RESUMEN

The study of flaviviruses, which cause some of the most important emerging tropical and sub-tropical human arbovirus diseases, has greatly benefited from the use of reverse genetic systems since its first development for yellow fever virus in 1989. Reverse genetics technology has completely revolutionized the study of these viruses, making it possible to manipulate their genomes and evaluate the direct effects of these changes on their biology and pathogenesis. The most commonly used reverse genetics system is the infectious clone technology. Whilst flavivirus infectious clones provide a powerful tool, their construction as full-length cDNA molecules in bacterial vectors can be problematic, laborious and time consuming, because they are often unstable, contain unwanted induced substitutions and may be toxic for bacteria due to viral protein expression. The incredible technological advances that have been made during the past 30years, such as the use of PCR or new sequencing methods, have allowed the development of new approaches to improve preexisting systems or elaborate new strategies that overcome these problems. This review summarizes the evolution and major technical breakthroughs in the development of flavivirus reverse genetics technologies and their application to the further understanding and control of these viruses and their diseases.


Asunto(s)
Flavivirus/genética , Genética Inversa , Clonación Molecular , ADN Complementario , Flavivirus/inmunología , Flavivirus/fisiología , Vectores Genéticos , ARN Viral/genética , Vacunas Virales/genética , Replicación Viral/genética
19.
PLoS One ; 10(9): e0138703, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26407018

RESUMEN

Isolation of viral pathogens from clinical and/or animal samples has traditionally relied on either cell cultures or laboratory animal model systems. However, virus viability is notoriously susceptible to adverse conditions that may include inappropriate procedures for sample collection, storage temperature, support media and transportation. Using our recently described ISA method, we have developed a novel procedure to isolate infectious single-stranded positive-sense RNA viruses from clinical or animal samples. This approach, that we have now called "ISA-lation", exploits the capacity of viral cDNA subgenomic fragments to re-assemble and produce infectious viral RNA in susceptible cells. Here, it was successfully used to rescue enterovirus, Chikungunya and Tick-borne encephalitis viruses from a variety of inactivated animal and human samples. ISA-lation represents an effective option to rescue infectious virus from clinical and/or animal samples that may have deteriorated during the collection and storage period, but also potentially overcomes logistic and administrative difficulties generated when complying with current health and safety and biosecurity guidelines associated with shipment of infectious viral material.


Asunto(s)
Virus ARN/genética , Virus ARN/aislamiento & purificación , ARN Viral/genética , Animales , Línea Celular , Humanos , Ratones
20.
Genome Announc ; 1(1)2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23469339

RESUMEN

Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People's Democratic Republic (PDR) (Laos).

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