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1.
Immunobiology ; 225(3): 151955, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517882

RESUMO

SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Pandemias , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/química , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/patogenicidade , Biologia Computacional/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Progressão da Doença , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Plasmídeos/química , Plasmídeos/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Conformação Proteica , Genética Reversa/métodos , Alinhamento de Sequência , Vacinas de Subunidades , Proteínas Virais/genética , Proteínas Virais/imunologia
2.
Nature ; 582(7813): 561-565, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32365353

RESUMO

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.


Assuntos
Betacoronavirus/genética , Clonagem Molecular/métodos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Genômica/métodos , Pneumonia Viral/virologia , Genética Reversa/métodos , Biologia Sintética/métodos , Animais , China/epidemiologia , Chlorocebus aethiops , Cromossomos Artificiais de Levedura/metabolismo , Infecções por Coronavirus/epidemiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Humanos , Mutação , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Vírus Sinciciais Respiratórios/genética , Saccharomyces cerevisiae/genética , Células Vero , Proteínas Virais/metabolismo , Zika virus/genética
3.
Arch Virol ; 165(5): 1079-1087, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144546

RESUMO

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.


Assuntos
Vírus da Doença Hemorrágica Epizoótica/genética , Genética Reversa/métodos , Virologia/métodos , Animais , Linhagem Celular , DNA Complementar/genética , Vírus da Doença Hemorrágica Epizoótica/crescimento & desenvolvimento , Mesocricetus , Plasmídeos , RNA Viral/genética , Recombinação Genética , Infecções por Reoviridae/virologia
4.
Virol J ; 16(1): 151, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31805959

RESUMO

BACKGROUND: Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. RESULT: In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. CONCLUSION: In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.


Assuntos
Vírus Bluetongue/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Genética Reversa/métodos , Virologia/métodos , Animais , Vírus Bluetongue/genética , Linhagem Celular , Cricetinae , Viabilidade Microbiana , Plasmídeos , Transfecção
5.
PLoS Biol ; 17(10): e3000502, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600204

RESUMO

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.


Assuntos
Vetores Aracnídeos/virologia , Abelhas/virologia , Evasão da Resposta Imune/genética , Vírus de RNA/genética , Varroidae/virologia , Animais , Abelhas/genética , Abelhas/imunologia , Abelhas/parasitologia , Células Clonais , Biblioteca Gênica , Variação Genética , Genótipo , Mutação , Filogenia , Interferência de RNA/imunologia , Vírus de RNA/classificação , Vírus de RNA/imunologia , Vírus de RNA/patogenicidade , Recombinação Genética , Genética Reversa/métodos , Seleção Genética , Virulência , Replicação Viral
6.
Virol J ; 16(1): 112, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488178

RESUMO

BACKGROUND: Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. METHODS: In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. RESULTS: The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 µg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. CONCLUSIONS: An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.


Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/genética , Avastrovirus/isolamento & purificação , Patos/virologia , Genética Reversa/métodos , Cultura de Vírus/métodos , Animais , Infecções por Astroviridae/virologia , Avastrovirus/crescimento & desenvolvimento , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Genoma Viral , Plasmídeos , RNA Viral/genética , Transfecção , Tropismo Viral , Vírion/genética
7.
Methods Mol Biol ; 2042: 185-204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385277

RESUMO

Chlamydia is a major etiological agent of human disease that affects millions of individuals worldwide. Historically, our understanding of the mechanisms that contribute to its pathogenesis has been limited. However, the recent development of powerful genetic tools for manipulating Chlamydia has resulted in significant gains in our ability to dissect its virulence mechanisms. These tools have overcome several barriers for manipulating intracellular pathogens and are amenable for the routine genetic engineering of Chlamydia. Here, we provide several detailed protocols for performing genetic analysis in Chlamydia trachomatis allowing investigators to elucidate how this obligate intracellular pathogen causes disease.


Assuntos
Chlamydia trachomatis/genética , DNA Bacteriano/genética , Genética Reversa/métodos , Animais , Infecções por Chlamydia/microbiologia , Chlorocebus aethiops , Ligação Genética , Humanos , Mutagênese , Polimorfismo de Nucleotídeo Único , Células Vero
8.
PLoS One ; 14(7): e0219168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276568

RESUMO

In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.


Assuntos
DNA Complementar/genética , Vírus da Caxumba/fisiologia , Mutação , Proteínas Virais/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Humanos , Vírus da Caxumba/genética , Genética Reversa/métodos , Inoculações Seriadas , Células Vero , Replicação Viral
9.
Virology ; 535: 227-231, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31325837

RESUMO

Mayaro virus (MAYV; family Togaviridae, genus Alphavirus) is an emerging global threat that can cause severe clinical manifestations similar to Zika, dengue, and chikungunya viruses. Currently, there is a lack of molecular tools to enable a better understanding of the transmission and pathogenesis of MAYV. Here, we detail the development and characterization of infectious clones of two strains of MAYV that produce infectious virus and replicate in mammalian and mosquito cells similarly to wild-type virus. Additionally, clone-derived viruses produced identical infection rates and phenotypes in CD-1 mice compared to the parental strains. This infectious clone system will provide a resource to the research community to analyze MAYV genetic determinants of virulence, determine vector competence, and develop vaccines.


Assuntos
Alphavirus/crescimento & desenvolvimento , Alphavirus/genética , DNA Complementar/genética , DNA Viral/genética , Genética Reversa/métodos , Vacinas Virais/isolamento & purificação , Alphavirus/imunologia , Alphavirus/patogenicidade , Infecções por Alphavirus/fisiopatologia , Infecções por Alphavirus/prevenção & controle , Animais , Linhagem Celular , Clonagem Molecular , Doenças Transmissíveis Emergentes/fisiopatologia , Doenças Transmissíveis Emergentes/prevenção & controle , Culicidae , DNA Complementar/isolamento & purificação , DNA Viral/isolamento & purificação , Humanos , Camundongos , Vacinologia/métodos , Vacinas Virais/genética , Vacinas Virais/imunologia , Virologia/métodos
10.
Virology ; 535: 130-135, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299489

RESUMO

Human astroviruses (HAstVs) are a frequent cause of gastroenteritis in young children and immunocompromised patients. The current report describes a new approach to recover genetically defined HAstVs through the use of a reverse genetics system based on a single DNA plasmid. This plasmid, carrying the full-length virus genome under a T7 promoter, is directly transfected into cells expressing T7 RNA polymerase, resulting in the rapid and robust recovery of infectious HAstV. The efficiency of the system was tested with the generation of a chimeric astrovirus having the HAstV serotype 1 replication machinery and the capsid derived from a HAstV serotype 8 virus. This new system provides an efficient and reproducible method to deepen our knowledge of astrovirus biology.


Assuntos
Mamastrovirus/crescimento & desenvolvimento , Mamastrovirus/genética , Genética Reversa/métodos , DNA Complementar/genética , Vetores Genéticos , Genoma Viral , Humanos , Plasmídeos
11.
Genetics ; 212(4): 1163-1179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243056

RESUMO

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.


Assuntos
Sistemas CRISPR-Cas , Malassezia/genética , Mutagênese , Genética Reversa/métodos , Agrobacterium/genética , Farmacorresistência Fúngica/genética , Deleção de Genes , Mutagênese Insercional , Transformação Genética
12.
Genetics ; 212(4): 1063-1073, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31243057

RESUMO

We develop a flexible and computationally efficient approach for analyzing high-throughput chemical genetic screens. In such screens, a library of genetic mutants is phenotyped in a large number of stresses. Typically, interactions between genes and stresses are detected by grouping the mutants and stresses into categories, and performing modified t-tests for each combination. This approach does not have a natural extension if mutants or stresses have quantitative or nonoverlapping annotations (e.g., if conditions have doses or a mutant falls into more than one category simultaneously). We develop a matrix linear model (MLM) framework that allows us to model relationships between mutants and conditions in a simple, yet flexible, multivariate framework. It encodes both categorical and continuous relationships to enhance detection of associations. We develop a fast estimation algorithm that takes advantage of the structure of MLMs. We evaluate our method's performance in simulations and in an Escherichia coli chemical genetic screen, comparing it with an existing univariate approach based on modified t-tests. We show that MLMs perform slightly better than the univariate approach when mutants and conditions are classified in nonoverlapping categories, and substantially better when conditions can be ordered in dosage categories. Therefore, it is an attractive alternative to current methods, and provides a computationally scalable framework for larger and complex chemical genetic screens. A Julia language implementation of MLMs and the code used for this paper are available at https://github.com/janewliang/GeneticScreen.jl and https://bitbucket.org/jwliang/mlm_gs_supplement, respectively.


Assuntos
Modelos Genéticos , Mutagênese , Genética Reversa/métodos , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Interação Gene-Ambiente , Modelos Lineares , Genética Reversa/normas , Software
13.
Virus Genes ; 55(4): 550-556, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161411

RESUMO

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Genética Reversa/métodos , Linhagem Celular , DNA Complementar , DNA Viral , Vírus da Encefalite Japonesa (Espécie)/ultraestrutura , Vetores Genéticos , Genoma Viral , Regiões Promotoras Genéticas , Sequenciamento Completo do Exoma
14.
Methods Mol Biol ; 2028: 27-84, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31228108

RESUMO

Antiviral RNA silencing and the resistance gene-conferred defense response are major antiviral immune systems in plants. Several of the components involved have been genetically or biochemically identified in Arabidopsis thaliana. One powerful tool to dissect antiviral immune systems involves a reverse genetic approach that analyzes Arabidopsis mutant lines with impaired antiviral defense responses. In particular, to better understand the signaling networks involved in the resistance gene-conferred antiviral response in host plants, establishment of mutant lines carrying the homozygous mutant allele and antiviral resistance gene is required. The information on well-characterized defense-related signaling mutant alleles and the PCR-based genotyping method provided in this chapter allows the efficient selection of Arabidopsis mutant lines that can be used to study antiviral resistance signaling networks and resistance mechanisms.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Resistência à Doença/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Genética Reversa , Transdução de Sinais , Arabidopsis/virologia , Cruzamentos Genéticos , Doenças das Plantas/virologia , Genética Reversa/métodos
15.
Viruses ; 11(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939777

RESUMO

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5' and 3' untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.


Assuntos
Reoviridae/crescimento & desenvolvimento , Reoviridae/genética , Genética Reversa/métodos , Animais , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Expressão Gênica , Genoma Viral , Corpos de Oclusão Virais , RNA Viral/genética , Células Sf9 , Spodoptera , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
J Virol Methods ; 265: 84-90, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615899

RESUMO

Reverse genetics systems are powerful tools for understanding the virulence mechanisms and gene functions of negative-sense RNA viruses. The reverse genetics systems commonly used for recombinant infectious hematopoietic necrosis virus (IHNV) are based on vaccinia virus infection. To avoid the potential biological safety risks associated with vaccinia virus, a recombinant IHNV virus strain Sn1203 (rIHNV-Sn1203) was rescued in this study using a mammalian cell line, BHK-21. The genome sequence authenticity of rIHNV-Sn1203 was confirmed using two silent genetic tags introduced by site-directed mutagenesis. Indirect immunofluorescence assays and transmission electron microscopy revealed that rIHNV-Sn1203 and wild-type IHNV-Sn1203 (wtIHNV-Sn1203) had identical immunogenicity and virion morphology. The virulence and pathogenicity of rIHNV-Sn1203 were assessed in vitro and in vivo. Although rIHNV-Sn1203 displayed trends toward delayed intracellular viral replication and lower virion yields compared with wtIHNV-Sn1203, statistical analyses revealed no significant differences between these two viruses. Moreover, rainbow trout challenged with rIHNV-Sn1203 and wtIHNV-Sn1203 showed indistinguishable mortality. Together, these results show that IHNV was successfully rescued using BHK-21 cells. This method is very convenient and may also be suitable for use in the recovery of other Novirhabdoviruses.


Assuntos
Vírus da Necrose Hematopoética Infecciosa/crescimento & desenvolvimento , Genética Reversa/métodos , Virologia/métodos , Animais , Linhagem Celular , Cricetinae , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Técnica Indireta de Fluorescência para Anticorpo , Vírus da Necrose Hematopoética Infecciosa/genética , Vírus da Necrose Hematopoética Infecciosa/patogenicidade , Vírus da Necrose Hematopoética Infecciosa/ultraestrutura , Microscopia Eletrônica de Transmissão , Oncorhynchus mykiss , Infecções por Rhabdoviridae/patologia , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/virologia , Análise de Sobrevida , Vírus Vaccinia/genética , Vírion/ultraestrutura , Replicação Viral
17.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 16-30, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30056088

RESUMO

Protein phosphatase 1 (PP1) catalyzes more than half of all phosphoserine/threonine dephosphorylation reactions in mammalian cells. In vivo PP1 does not exist as a free catalytic subunit but is always associated with at least one regulatory PP1-interacting protein (PIP) to generate a large set of distinct holoenzymes. Each PP1 complex controls the dephosphorylation of only a small subset of PP1 substrates. We screened the literature for genetically engineered mouse models and identified models for all PP1 isoforms and 104 PIPs. PP1 itself and at least 49 PIPs were connected to human disease-associated phenotypes. Additionally, phenotypes related to 17 PIPs were clearly linked to altered PP1 function, while such information was lacking for 32 other PIPs. We propose structural reverse genetics, which combines structural characterization of proteins with mouse genetics, to identify new PP1-related therapeutic targets. The available mouse models confirm the pleiotropic action of PP1 in health and diseases.


Assuntos
Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/fisiologia , Processamento Alternativo , Animais , Doença , Genótipo , Holoenzimas/metabolismo , Holoenzimas/fisiologia , Humanos , Camundongos , Modelos Animais , Fenótipo , Fosforilação , Isoformas de Proteínas , Genética Reversa/métodos , Especificidade por Substrato/fisiologia
18.
J Infect Dis ; 220(2): 187-194, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-30383246

RESUMO

BACKGROUND: Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers. METHODS: The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid. RESULTS: The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum. CONCLUSIONS: Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.


Assuntos
Infecções por Picornaviridae/virologia , Genética Reversa/métodos , Rhinovirus/genética , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Feminino , Humanos , Masculino , Muco , Infecções por Picornaviridae/transmissão , Rhinovirus/fisiologia
19.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463961

RESUMO

The cocirculation of H5N1 and H9N2 avian influenza viruses in birds in Egypt provides reassortment opportunities between these two viruses. However, little is known about the emergence potential of reassortants derived from Egyptian H5N1 and H9N2 viruses and about the biological properties of such reassortants. To evaluate the potential public health risk of reassortants of these viruses, we used reverse genetics to generate the 63 possible reassortants derived from contemporary Egyptian H5N1 and H9N2 viruses, containing the H5N1 surface gene segments and combinations of the H5N1 and H9N2 internal gene segments, and analyzed their genetic compatibility, replication ability, and virulence in mice. Genes in the reassortants showed remarkably high compatibility. The replication of most reassortants was higher than the parental H5N1 virus in human cells. Six reassortants were thought to emerge in birds under neutral or positive selective pressure, and four of them had higher pathogenicity in vivo than the parental H5N1 and H9N2 viruses. Our results indicated that H5N1-H9N2 reassortants could be transmitted efficiently to mammals with significant public health risk if they emerge in Egypt, although the viruses might not emerge frequently in birds.IMPORTANCE Close interaction between avian influenza (AI) viruses and humans in Egypt appears to have resulted in many of the worldwide cases of human infections by both H5N1 and H9N2 AI viruses. Egypt is regarded as a hot spot of AI virus evolution. Although no natural reassortant of H5N1 and H9N2 AI viruses has been reported so far, their cocirculation in Egypt may allow emergence of reassortants that may present a significant public health risk. Using reverse genetics, we report here the first comprehensive data showing that H5N1-N9N2 reassortants have fairly high genetic compatibility and possibly higher pathogenicity in mammals, including humans, than the parental viruses. Our results provide insight into the emergence potential of avian H5N1-H9N2 reassortants that may pose a high public health risk.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Vírus Reordenados/genética , Animais , Aves/genética , Cães , Genes Virais , Células HEK293 , Humanos , Influenza Aviária/virologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mamíferos/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Filogenia , Genética Reversa/métodos , Virulência , Replicação Viral
20.
Appl Microbiol Biotechnol ; 103(2): 869-880, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30535678

RESUMO

Acetaldehyde is synthesized by yeast during the main fermentation period of beer production, which causes an unpleasant off-flavor. Therefore, there has been extensive effort toward reducing acetaldehyde to obtain a beer product with better flavor and anti-staling ability. In this study, we discovered that acetaldehyde production in beer brewing is closely related with the intracellular NADH equivalent regulated by the citric acid cycle. However, there was no significant relationship between acetaldehyde production and amino acid metabolism. A reverse engineering strategy to increase the intracellular NADH/NAD+ ratio reduced the final acetaldehyde production level, and vice versa. This work offers new insight into acetaldehyde metabolism and further provides efficient strategies for reducing acetaldehyde production by the regulating the intracellular NADH/NAD+ ratio through cofactor engineering.


Assuntos
Acetaldeído/metabolismo , Cerveja/microbiologia , Engenharia Metabólica/métodos , NAD/metabolismo , Genética Reversa/métodos , Saccharomyces/genética , Saccharomyces/metabolismo , Fermentação
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