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1.
J Immunol ; 206(7): 1653-1667, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33637616

RESUMO

The reptile MHC class I (MCH-I) and MHC class II proteins are the key molecules in the immune system; however, their structure has not been investigated. The crystal structure of green anole lizard peptide-MHC-I-ß2m (pMHC-I or pAnca-UA*0101) was determined in the current study. Subsequently, the features of pAnca-UA*0101 were analyzed and compared with the characteristics of pMHC-I of four classes of vertebrates. The amino acid sequence identities between Anca-UA*0101 and MHC-I from other species are <50%; however, the differences between the species were reflected in the topological structure. Significant characteristics of pAnca-UA*0101 include a specific flip of ∼88° and an upward shift adjacent to the C terminus of the α1- and α2-helical regions, respectively. Additionally, the lizard MHC-I molecule has an insertion of 2 aa (VE) at positions 55 and 56. The pushing force from 55-56VE triggers the flip of the α1 helix. Mutagenesis experiments confirmed that the 55-56VE insertion in the α1 helix enhances the stability of pAnca-UA*0101. The peptide presentation profile and motif of pAnca-UA*0101 were confirmed. Based on these results, the proteins of three reptile lizard viruses were used for the screening and confirmation of the candidate epitopes. These data enhance our understanding of the systematic differences between five classes of vertebrates at the gene and protein levels, the formation of the pMHC-I complex, and the evolution of the MHC-I system.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Lagartos/imunologia , Infecções por Nidovirales/imunologia , Nidovirales/fisiologia , Proteínas de Répteis/química , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Cristalografia por Raios X , Epitopos/genética , Evolução Molecular , Antígenos de Histocompatibilidade Classe I/genética , Sistema Imunitário , Imunidade , Filogenia , Polimorfismo Genético , Conformação Proteica , Estabilidade Proteica , Proteínas de Répteis/genética
2.
J Gen Virol ; 103(5)2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35579608

RESUMO

The purification of virus particles is an essential process for the manufacture of vaccines. However, the application of different purification processes may affect the quality of the virus particles, such as structural integrity and homogeneity, which may further influence the infectivity and immunogenicity of the purified virus. In this study, we took Feline calicivirus (FCV), a common natural pathogen in cats belonging to Caliciviridae, as a research model. By using cryo-electron microscopy (cryo-EM), we incorporated the 3D classification process as a virus flexibility evaluation system. Cryo-EM images of virus particles resulting from different purification processes were compared at near-atomic resolution. The results indicated that molecular sieving purification will impact the stability of P-domains through increasing flexibility as determined by the evaluation system, which can be extended to assess the purification effect on the entire particle. This evaluation process can be further applied to all non-enveloped viruses.


Assuntos
Infecções por Caliciviridae , Caliciviridae , Calicivirus Felino , Doenças do Gato , Vírus , Animais , Infecções por Caliciviridae/veterinária , Gatos , Microscopia Crioeletrônica/métodos , Vírion/química
3.
J Immunol ; 204(1): 147-158, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31776204

RESUMO

The African clawed frog, Xenopus laevis, is a model species for amphibians. Before metamorphosis, tadpoles do not efficiently express the single classical MHC class I (MHC-I) molecule Xela-UAA, but after metamorphosis, adults express this molecule in abundance. To elucidate the Ag-presenting mechanism of Xela-UAA, in this study, the Xela-UAA structure complex (pXela-UAAg) bound with a peptide from a synthetic random peptide library was determined. The amino acid homology between the Xela-UAA and MHC-I sequences of different species is <45%, and these differences are fully reflected in the three-dimensional structure of pXela-UAAg. Because of polymorphisms and interspecific differences in amino acid sequences, pXela-UAAg forms a distinct peptide-binding groove and presents a unique peptide profile. The most important feature of pXela-UAAg is the two-amino acid insertion in the α2-helical region, which forms a protrusion of ∼3.8 Å that is involved in TCR docking. Comparison of peptide-MHC-I complex (pMHC-I) structures showed that only four amino acids in ß2-microglobulin that were bound to MHC-I are conserved in almost all jawed vertebrates, and the most unique feature in nonmammalian pMHC-I molecules is that the AB loop bound ß2-microglobulin. Additionally, the binding distance between pMHC-I and CD8 molecules in nonmammals is different from that in mammals. These unique features of pXela-UAAg provide enhanced knowledge of T cell immunity and bridge the knowledge gap regarding the coevolutionary progression of the MHC-I complex from aquatic to terrestrial species.


Assuntos
Genes MHC Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Xenopus laevis/imunologia , Animais , Genes MHC Classe I/genética , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Xenopus laevis/genética
4.
J Immunol ; 202(12): 3493-3506, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31076531

RESUMO

Bats are natural reservoir hosts, harboring more than 100 viruses, some of which are lethal to humans. The asymptomatic coexistence with viruses is thought to be connected to the unique immune system of bats. MHC class I (MHC I) presentation is closely related to cytotoxic lymphocyte immunity, which plays an important role in viral resistance. To investigate the characteristics of MHC I presentation in bats, the crystal structures of peptide-MHC I complexes of Pteropus alecto, Ptal-N*01:01/HEV-1 (DFANTFLP) and Ptal-N*01:01/HEV-2 (DYINTNLVP), and two related mutants, Ptal-N*01:01/HEV-1PΩL (DFANTFLL) and Ptal-N*01:01ΔMDL/HEV-1, were determined. Through structural analysis, we found that Ptal-N*01:01 had a multi-Ala-assembled pocket B and a flexible hydrophobic pocket F, which could accommodate variable anchor residues and allow Ptal-N*01:01 to bind numerous peptides. Three sequential amino acids, Met, Asp, and Leu, absent from the α1 domain of the H chain in other mammals, were present in this domain in the bat. Upon deleting these amino acids and determining the structure in p/Ptal-N*01:01ΔMDL/HEV-1, we found they helped form an extra salt-bridge chain between the H chain and the N-terminal aspartic acid of the peptide. By introducing an MHC I random peptide library for de novo liquid chromatography-tandem mass spectrometry analysis, we found that this insertion module, present in all types of bats, can promote MHC I presentation of peptides with high affinity during the peptide exchange process. This study will help us better understand how bat MHC I presents high-affinity peptides from an extensive binding peptidome and provides a foundation to understand the cellular immunity of bats.


Assuntos
Antígenos/metabolismo , Quirópteros/imunologia , Cristalografia por Raios X/métodos , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Animais , Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Imunidade Celular , Espectrometria de Massas , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica
5.
BMC Genomics ; 20(1): 45, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646841

RESUMO

BACKGROUND: Taste and aroma, which are important organoleptic qualities of apricot (Prunus armeniaca L.) fruit, undergo rapid and substantial changes during ripening. However, the associated molecular mechanisms remain unclear. The goal of this study was to identify candidate genes for flavor compound metabolism and to construct a regulatory transcriptional network. RESULTS: We characterized the transcriptome of the 'Jianali' apricot cultivar, which exhibits substantial changes in flavor during ripening, at 50 (turning), 73 (commercial maturation) and 91 (full ripe) days post anthesis (DPA) using RNA sequencing (RNA-Seq). A weighted gene co-expression network analysis (WGCNA) revealed that four of 19 modules correlated highly with flavor compound metabolism (P < 0.001). From them, we identified 1237 differentially expressed genes, with 16 intramodular hubs. A proposed pathway model for flavor compound biosynthesis is presented based on these genes. Two SUS1 genes, as well as SPS2 and INV1 were correlated with sugar biosynthesis, while NADP-ME4, two PK-like and mitochondrial energy metabolism exerted a noticeable effect on organic acid metabolism. CCD1 and FAD2 were identified as being involved in apocarotenoid aroma volatiles and lactone biosynthesis, respectively. Five sugar transporters (Sweet10, STP13, EDR6, STP5.1, STP5.2), one aluminum-activated malate transporter (ALMT9) and one ABCG transporter (ABCG11) were associated with the transport of sugars, organic acids and volatiles, respectively. Sixteen transcription factors were also highlighted that may also play regulatory roles in flavor quality development. CONCLUSIONS: Apricot RNA-Seq data were obtained and used to generate an annotated set of predicted expressed genes, providing a platform for functional genomic research. Using network analysis and pathway mapping, putative molecular mechanisms for changes in apricot fruit taste and aroma during ripening were elucidated.


Assuntos
Frutas/crescimento & desenvolvimento , Frutas/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Prunus armeniaca/genética , Olfato , Paladar , Vias Biossintéticas/genética , Frutas/fisiologia , Perfilação da Expressão Gênica , Genes de Plantas , Reguladores de Crescimento de Plantas/biossíntese , Transdução de Sinais/genética , Solubilidade , Açúcares/metabolismo , Transcriptoma/genética , Compostos Orgânicos Voláteis/metabolismo
6.
Immunogenetics ; 71(5-6): 395-405, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30941483

RESUMO

Major histocompatibility complex (MHC) class I molecules play a crucial role in the immune response by binding and presenting pathogen-derived peptides to specific CD8+ T cells. From cDNA of 20 individuals of wild grass carp (Ctenopharyngodon idellus), we could amplify one or two alleles each of classical MHC class I genes Ctid-UAA and Ctid-UBA. In total, 27 and 22 unique alleles of Ctid-UAA and Ctid-UBA were found. The leader, α1, transmembrane and cytoplasmic regions distinguish between Ctid-UAA and Ctid-UBA, and their encoded α1 domain sequences belong to the ancient lineages α1-V and α1-II, respectively, which separated several hundred million years ago. However, Ctid-UAA and Ctid-UBA share allelic lineage variation in their α2 and α3 sequences, in a pattern suggestive of past interlocus recombination events that transferred α2+α3 fragments. The allelic Ctid-UAA and Ctid-UBA variation involves ancient variation between domain lineages α2-I and α2-II, which in the present study was dated back to before the ancestral separation of teleost fish and spotted gar (> 300 million years ago). This is the first report with compelling evidence that recombination events combining different ancient α1 and α2 domain lineages had a major impact on the allelic variation of two different classical MHC class I genes within the same species.


Assuntos
Alelos , Carpas/genética , Carpas/imunologia , Genes MHC Classe I , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Sequência de Aminoácidos , Animais , Carpas/classificação , Evolução Molecular , Ligação Genética , Loci Gênicos , Genoma , Genômica/métodos , Modelos Moleculares , Conformação Molecular , Filogenia , Polimorfismo Genético , Recombinação Genética , Relação Estrutura-Atividade
7.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29263258

RESUMO

Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag positions 40 to 48; RMANVSTGR [RMA9]). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular-dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and in vitro refolding of the mutant FLA-E*01801 complex demonstrated that Glu63 and Trp167 in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine.IMPORTANCE Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC-I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp167 blocks the left side of pocket A, causing P1D to conflict with Glu63 We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We identified 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC-I molecules present peptides.


Assuntos
Produtos do Gene gag/química , Antígenos de Histocompatibilidade Classe I/química , Vírus da Imunodeficiência Felina/química , Peptídeos/química , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Motivos de Aminoácidos , Animais , Apresentação de Antígeno , Sítios de Ligação , Gatos , Cristalografia por Raios X , Produtos do Gene gag/imunologia , HIV-1/química , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Vírus da Imunodeficiência Felina/imunologia , Peptídeos/imunologia
8.
Proteomics ; 17(23-24)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29052333

RESUMO

Significant differences exist between the highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) and its attenuated pathogenic (AP) strain in the ability to infect host cells. The mechanisms by which different virulent strains invade host cells remain relatively unknown. In this study, pulmonary alveolar macrophages (PAMs) are infected with HP-PRRSV (HuN4) and AP-PRRSV (HuN4-F112) for 24 h, then harvested and subjected to label-free quantitative MS. A total of 2849 proteins are identified, including 95 that are differentially expressed. Among them, 26 proteins are located on the membrane. The most differentially expressed proteins are involved in response to stimulus, metabolic process, and immune system process, which mainly have the function of binding and catalytic activity. Cluster of differentiation CD163, vimentin (VIM), and nmII as well as detected proteins are assessed together by string analysis, which elucidated a potentially different infection mechanism. According to the function annotations, PRRSV with different virulence may mainly differ in immunology, inflammation, immune evasion as well as cell apoptosis. This is the first attempt to explore the differential characteristics between HP-PRRSV and its attenuated PRRSV infected PAMs focusing on membrane proteins which will be of great help to further understand the different infective mechanisms of HP-PRRSV and AP-PRRSV.


Assuntos
Macrófagos Alveolares/metabolismo , Proteínas de Membrana/análise , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Proteoma/análise , Proteômica/métodos , Alvéolos Pulmonares/metabolismo , Animais , Células Cultivadas , Interações Hospedeiro-Patógeno , Macrófagos Alveolares/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Alvéolos Pulmonares/virologia , Suínos , Virulência
9.
Commun Biol ; 7(1): 586, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755285

RESUMO

Bats serve as reservoirs for numerous zoonotic viruses, yet they typically remain asymptomatic owing to their unique immune system. Of particular significance is the MHC-I in bats, which plays crucial role in anti-viral response and exhibits polymorphic amino acid (AA) insertions. This study demonstrated that both 5AA and 3AA insertions enhance the thermal stability of the bat MHC-I complex and enrich the diversity of bound peptides in terms of quantity and length distribution, by stabilizing the 310 helix, a region prone to conformational changes during peptide loading. However, the mismatched insertion could diminish the stability of bat pMHC-I. We proposed that a suitable insertion may help bat MHC-I adapt to high body temperatures during flight while enhancing antiviral responses. Moreover, this site-specific insertions may represent a strategy of evolutionary adaptation of MHC-I molecules to fluctuations in body temperature, as similar insertions have been found in other lower vertebrates.


Assuntos
Quirópteros , Antígenos de Histocompatibilidade Classe I , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Estabilidade Proteica , Peptídeos/química , Peptídeos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Apresentação de Antígeno , Mutagênese Insercional
10.
Biomedicines ; 9(9)2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34572339

RESUMO

(1) Background: The cholinergic anti-inflammatory pathway (CAP) has been implicated in the regulation of various diseases, including chronic inflammatory cardiovascular disorders such as atherosclerosis (AS). This study aims to explore the underlying regulatory mechanisms of CAP activity in the progression of AS. (2) Methods: The Apoe-/- mice were subjected to sham, bilateral cervical vagotomy surgery (VGX), and VGX supplemented with Gainesville Tokushima scientists (GTS)-21 (4 mg/kg/d) and then fed with a high-fat diet for 10 weeks. Atherosclerotic lesion size and inflammation levels were investigated by histology and inflammatory cytokines analysis. The blood M1/M2 macrophages were analyzed by flow cytometry. Primary mouse bone marrow-derived macrophages (BMDM), peritoneal macrophages, and RAW264.7 cells were treated with CAP agonists acetylcholine (Ach) and GTS-21 to study their effects on macrophage functions. (3) Results: Compared with the sham group, inhibition of CAP by the VGX resulted in growing aortic lipid plaque area, deteriorated inflammatory levels, and aberrant quantity of M1/M2 macrophages in Apoe-/- mice. However, these detrimental effects of VGX were significantly ameliorated by the reactivation of CAP through GTS-21 treatment. The in vitro study using macrophages revealed that stimulation with CAP agonists suppressed M1, but promoted M2 macrophage polarization through the upregulation of TNFAIP3 and phosphorylation STAT3 levels, respectively. Moreover, the activation of CAP inhibited the formation of macrophage foam cells in the peritoneal cavity by regulating genes related to cholesterol metabolism. (4) Conclusions: This study provides novel evidence and mechanisms that the CAP plays an important role in the regulation of AS development by controlling macrophage functions, implying a potential use of CAP activation as a therapeutic strategy for AS treatment.

11.
Front Immunol ; 12: 592447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717070

RESUMO

The micropolymorphism of major histocompatibility complex class I (MHC-I) can greatly alter the plasticity of peptide presentation, but elucidating the underlying mechanism remains a challenge. Here we investigated the impact of the micropolymorphism on peptide presentation of swine MHC-I (termed swine leukocyte antigen class I, SLA-I) molecules via immunopeptidomes that were determined by our newly developed random peptide library combined with the mass spectrometry (MS) de novo sequencing method (termed RPLD-MS) and the corresponding crystal structures. The immunopeptidomes of SLA-1*04:01, SLA-1*13:01, and their mutants showed that mutations of residues 156 and 99 could expand and narrow the ranges of peptides presented by SLA-I molecules, respectively. R156A mutation of SLA-1*04:01 altered the charge properties and enlarged the volume size of pocket D, which eliminated the harsh restriction to accommodate the third (P3) anchor residue of the peptide and expanded the peptide binding scope. Compared with 99Tyr of SLA-1*0401, 99Phe of SLA-1*13:01 could not form a conservative hydrogen bond with the backbone of the P3 residues, leading to fewer changes in the pocket properties but a significant decrease in quantitative of immunopeptidomes. This absent force could be compensated by the salt bridge formed by P1-E and 170Arg. These data illustrate two distinguishing manners that show how micropolymorphism alters the peptide-binding plasticity of SLA-I alleles, verifying the sensitivity and accuracy of the RPLD-MS method for determining the peptide binding characteristics of MHC-I in vitro and helping to more accurately predict and identify MHC-I restricted epitopes.


Assuntos
Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia Líquida , Dicroísmo Circular , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Mutação , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/imunologia , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Suínos , Espectrometria de Massas em Tandem , Difração de Raios X
12.
PLoS One ; 12(2): e0172270, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28241064

RESUMO

BACKGROUND: Brain derived neurotrophic factor (BDNF) is one of the most important regulatory proteins in the pathophysiology of major depressive disorder (MDD). Increasing numbers of studies have reported the relationship between serum/plasma BDNF and antidepressants (ADs). However, the potential effects of several classes of antidepressants on BDNF concentrations are not well known. Hence, our meta-analyses aims to review the effects of differential antidepressant drugs on peripheral BDNF levels in MDD and make some recommendations for future research. METHODS: Electronic databases including PubMed, EMBASE, the Cochrane Library, Web of Science, and PsycINFO were searched from 1980 to June 2016. The change in BDNF levels were compared between baseline and post-antidepressants treatment by use of the standardized mean difference (SMD) with 95% confidence intervals (CIs). All statistical tests were two-sided. RESULTS: We identified 20 eligible trials of antidepressants treatments for BDNF in MDD. The overall effect size for all drug classes showed that BDNF levels were elevated following a course of antidepressants use. For between-study heterogeneity by stratification analyses, we detect that length of treatment and blood samples are significant effect modifiers for BDNF levels during antidepressants treatment. While both SSRIs and SNRIs could increase the BDNF levels after a period of antidepressant medication treatment, sertraline was superior to other three drugs (venlafaxine, paroxetine or escitalopram) in the early increase of BDNF concentrations with SMD 0.53(95% CI = 0.13-0.93; P = 0.009). CONCLUSIONS: There is some evidence that treatment of antidepressants appears to be effective in the increase of peripheral BDNF levels. More robust evidence indicates that different types of antidepressants appear to induce differential effects on the BDNF levels. Since sertraline makes a particular effect on BDNF concentration within a short amount of time, there is potential value in exploring its relationship with BDNF and its pharmacological mechanism concerning peripheral blood BDNF. Further confirmatory trials are required for both observations.


Assuntos
Antidepressivos/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/sangue , Depressão/sangue , Depressão/tratamento farmacológico , Citalopram/uso terapêutico , Ensaios Clínicos como Assunto , Interpretação Estatística de Dados , Humanos , Paroxetina/uso terapêutico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sertralina/uso terapêutico , Cloridrato de Venlafaxina/uso terapêutico
13.
Res Vet Sci ; 107: 132-140, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27473986

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) has a condensed single-stranded positive-sense RNA genome that contains several overlapping regions. The transcription regulatory sequence (TRS) is the important cis-acting element participating in PRRSV discontinuous transcription process. Based on reverse genetic system of type 2 highly pathogenic PRRSV cell-passage attenuated strain pHuN4-F112, firefly luciferase or Renilla luciferase genes were inserted between ORF1b and ORF2. An extra TRS6 was embedded behind the foreign luciferase genes. pA-Fluc and pA-Rluc were constructed and successfully rescued in MARC-145 cells. The phenotypical characteristics of the progeny virus were indistinguishable from those of vHuN4-F112 and were genetically stable for at least 25 cell passages. Mutant virus-infected cells were lysed at different time points to assess luciferase activities and measure foreign gene expression levels. The results showed identical variations in the luciferase activities of the recombinants in MARC-145 cells, indicating that they were suitable for monitoring viral propagation in PRRSV-permissive cell cultures. They were also used to infect pulmonary alveolar macrophages, which yielded similar variations in luciferase activities. Therefore, vA-Fluc and vA-Rluc present powerful new tools to monitor PRRSV propagation in both passaged and target cells.


Assuntos
Luciferases/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína , Proteínas Recombinantes/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Luciferases/genética , Macrófagos Alveolares/virologia , Proteínas Recombinantes/genética , Suínos
14.
Sci Rep ; 5: 17010, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26581169

RESUMO

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5' untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5' UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies.


Assuntos
MicroRNAs/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Replicação Viral , Regiões 5' não Traduzidas/genética , Administração Intranasal , Animais , Antivirais/farmacologia , Sequência de Bases , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Interferon-alfa/genética , Interferon-alfa/metabolismo , MicroRNAs/administração & dosagem , Dados de Sequência Molecular , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sus scrofa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral/efeitos dos fármacos
15.
Vet Microbiol ; 179(3-4): 242-9, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26162970

RESUMO

Three porcine reproductive and respiratory syndrome viruses (PRRSV), NT1, NT2, and NT3, were isolated from three dying piglets from a single pig farm in Jiangsu Province, China. Whole genome sequencing revealed that the three isolates share the highest homology with JXA1-P80, an attenuated vaccine strain developed by serial passage of highly pathogenic PRRSV JXA1 in MARC-145 cells. More than ten amino acids residues in ORF1a, ORF1b, GP4, and GP5 that were thought to be unique to JXA1 attenuated on MARC-145 cells were each found in the corresponding locations of NT1, NT2, and NT3. In virulence assays, piglets infected with NT1, NT2, or NT3 exhibited clinical signs of disease, including high fever, anorexia, and respiratory distress, leading to the death of the majority of the piglets within two weeks. Collectively, these data indicate that NT1, NT2, and NT3 are highly pathogenic PRRSVs and they are likely to be revertants of the vaccine strain JXA1-P80.


Assuntos
Genoma Viral/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vacinas Virais/genética , Animais , Sequência de Bases , China , Dados de Sequência Molecular , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Análise de Sequência de DNA/veterinária , Inoculações Seriadas , Especificidade da Espécie , Suínos , Virulência
16.
Science ; 327(5968): 1018-21, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20167789

RESUMO

A range of applications, from predicting the spread of human and electronic viruses to city planning and resource management in mobile communications, depend on our ability to foresee the whereabouts and mobility of individuals, raising a fundamental question: To what degree is human behavior predictable? Here we explore the limits of predictability in human dynamics by studying the mobility patterns of anonymized mobile phone users. By measuring the entropy of each individual's trajectory, we find a 93% potential predictability in user mobility across the whole user base. Despite the significant differences in the travel patterns, we find a remarkable lack of variability in predictability, which is largely independent of the distance users cover on a regular basis.


Assuntos
Comportamento , Telefone Celular , Atividades Humanas , Viagem , Previsões , Humanos , Locomoção , Modelos Estatísticos , Probabilidade
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