RESUMO
Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Glicólise , Nicotiana/enzimologia , Tombusvirus/fisiologia , Replicação Viral/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Interações entre Hospedeiro e Microrganismos , Nicotiana/metabolismo , Nicotiana/virologia , Tombusvirus/genética , Tombusvirus/metabolismoRESUMO
Significant phenotypic heterogeneity exists in patients with all subtypes of myeloproliferative neoplasms (MPN), including essential thrombocythaemia (ET). Single-cell RNA sequencing (scRNA-Seq) holds the promise of unravelling the biology of MPN at an unprecedented level of resolution. Herein we employed this approach to dissect the transcriptomes in the CD34+ cells from the peripheral blood of seven previously untreated ET patients and one healthy adult. The mutational profiles in these patients were as follows: JAK2 V617F in two, CALR in three (one type I and two type II) and triple-negative (TN) in two. Our results reveal substantial heterogeneity within this enrolled cohort of patients. Activation of JAK/STAT signalling was recognized in discrepant progenitor lineages among different samples. Significantly disparate molecular profiling was identified in the comparison between ET patients and the control, between patients with different driver mutations (JAK2 V617F and CALR exon 9 indel), and even between patients harbouring the same driver. Intra-individual clonal diversity was also found in the CD34+ progenitor population of a patient, possibly indicating the presence of multiple clones in this case. Estimation of subpopulation size based on cellular immunophenotyping suggested differentiation bias in all analysed samples. Furthermore, combining the transcriptomic information with data from targeted sequencing enabled us to unravel key somatic mutations that are molecularly relevant. To conclude, we demonstrated that scRNA-Seq extended our knowledge of clonal diversity and inter-individual heterogeneity in patients with ET. The obtained information could potentially leapfrog our efforts in the elucidation of the pathogenesis of the disease.
Assuntos
Calreticulina , Janus Quinase 2 , RNA-Seq , Análise de Célula Única , Trombocitemia Essencial , Transcriptoma , Adulto , Substituição de Aminoácidos , Calreticulina/genética , Calreticulina/metabolismo , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Trombocitemia Essencial/sangue , Trombocitemia Essencial/genéticaRESUMO
Viruses accomplish their replication by exploiting many cellular resources, including metabolites and energy. Similarly to other (+)RNA viruses, tomato bushy stunt virus (TBSV) induces major changes in infected cells. However, the source of energy required to fuel TBSV replication is unknown. We find that TBSV co-opts the cellular glycolytic ATP-generating pyruvate kinase (PK) directly into the viral replicase complex to boost progeny RNA synthesis. The co-opted PK generates high levels of ATP within the viral replication compartment at the expense of a reduction in cytosolic ATP pools. The ATP generated by the co-opted PK is used to promote the helicase activity of recruited cellular DEAD-box helicases, which are involved in the production of excess viral (+)RNA progeny. Altogether, recruitment of PK and local production of ATP within the replication compartment allow the virus replication machinery an access to plentiful ATP, facilitating robust virus replication.
Assuntos
Trifosfato de Adenosina/metabolismo , Glicólise/fisiologia , Piruvato Quinase/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Tombusvirus/metabolismo , Replicação Viral/fisiologia , RNA Helicases DEAD-box/metabolismo , Escherichia coli , Técnicas de Silenciamento de Genes , Inativação Gênica , Interações Hospedeiro-Patógeno/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Plasmídeos , Proteômica , Vírus de RNA/enzimologia , Vírus de RNA/genética , Vírus de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virologia , Tombusvirus/enzimologia , Tombusvirus/genética , Replicação Viral/genéticaRESUMO
The intricate interactions between viruses and hosts include exploitation of host cells for viral replication by using many cellular resources, metabolites and energy. Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, induces major changes in infected cells that lead to the formation of large replication compartments consisting of aggregated peroxisomal and ER membranes. Yet, it is not known how TBSV obtains the energy to fuel these energy-consuming processes. In the current work, the authors discovered that TBSV co-opts the glycolytic ATP-generating Pgk1 phosphoglycerate kinase to facilitate the assembly of new viral replicase complexes. The recruitment of Pgk1 into the viral replication compartment is through direct interaction with the viral replication proteins. Altogether, we provide evidence that the ATP generated locally within the replication compartment by the co-opted Pgk1 is used to fuel the ATP-requirement of the co-opted heat shock protein 70 (Hsp70) chaperone, which is essential for the assembly of new viral replicase complexes and the activation of functional viral RNA-dependent RNA polymerase. The advantage of direct recruitment of Pgk1 into the virus replication compartment could be that the virus replicase assembly does not need to intensively compete with cellular processes for access to ATP. In addition, local production of ATP within the replication compartment could greatly facilitate the efficiency of Hsp70-driven replicase assembly by providing high ATP concentration within the replication compartment.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Fosfoglicerato Quinase/metabolismo , Tombusvirus/crescimento & desenvolvimento , Montagem de Vírus/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Saccharomyces cerevisiae , Nicotiana/virologiaRESUMO
Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly.
Assuntos
Arabidopsis/enzimologia , Interações Hospedeiro-Patógeno , RNA Polimerase Dependente de RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Tombusvirus/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Replicação Viral , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Deleção de Genes , Teste de Complementação Genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5' end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication.
Assuntos
RNA Helicases DEAD-box/genética , Instabilidade Genômica/genética , Nicotiana/virologia , RNA Viral/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tombusvirus/genética , Adenosina Trifosfatases/metabolismo , Exorribonucleases/metabolismo , Vetores Genéticos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Nicotiana/genética , Replicação Viral/genéticaRESUMO
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the 70 kDa heat-shock protein (Hsp70). Heat-shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is constitutively expressed; thus, it may be expressed under physiological conditions. In C6/36 cells, Hsc70 is upregulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at the late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C and D; of these, isoform D helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Assuntos
Clatrina/metabolismo , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/metabolismo , Endocitose/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Animais , Linhagem Celular , Cricetinae , Culicidae/virologia , Encefalite Japonesa/virologia , Dados de Sequência Molecular , Isoformas de Proteínas , Internalização do VírusRESUMO
Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.
Assuntos
Sistema Nervoso Central/virologia , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/genética , Recombinação Genética , Animais , Sistema Nervoso Central/fisiopatologia , Culicidae/virologia , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/patologia , Encefalite Japonesa/transmissão , Encefalite Japonesa/virologia , Humanos , RNA Viral/genética , Replicação ViralRESUMO
RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.
Assuntos
Retículo Endoplasmático/metabolismo , Fosfatidato Fosfatase/metabolismo , Vírus de RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação Viral/fisiologia , Western Blotting , Retículo Endoplasmático/genética , Microscopia Confocal , Fosfatidato Fosfatase/genética , Vírus de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Nicotiana/virologiaRESUMO
Japanese encephalitis (JE) virus is the most common cause of epidemic viral encephalitis in the world. The virus mainly infects neuronal cells and causes an inflammatory response after invasion of the parenchyma of the brain. The death of neurons is frequently observed, in which demyelinated axons are commonly seen. The mechanism that accounts for the occurrence of demyelination is ambiguous thus far. With a mouse model, the present study showed that myelin-specific antibodies appeared in sera, particularly in those mice with evident symptoms. Meanwhile, specific T cells proliferating in response to stimulation by myelin basic protein (MBP) was also shown in these mice. Taken together, our results suggest that autoimmunity may play an important role in the destruction of components, e.g., MBP, of axon-surrounding myelin, resulting in demyelination in the mouse brain after infection with the JE virus.
Assuntos
Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/imunologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/virologia , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Animais , Anticorpos Antivirais/imunologia , Apoptose , Encéfalo/patologia , Encéfalo/virologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/patologia , Encefalite Japonesa/patologia , Feminino , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , Proteína Básica da Mielina/metabolismo , Necrose , Linfócitos T/imunologiaRESUMO
Dengue viruses (DENVs) generally induce apoptosis in mammalian cells but cause only minor damage in mosquito cells. To find genes involved in determining the cell fate, datasets derived from expressed sequence tags (ESTs) of C6/36 cells with and without infection were established. Of overexpressed genes found in infected dataset, chaperone proteins were validated significantly upregulated in C6/36 cells at 24 hpi. It suggests that DENV-2 in mosquito cells activates the unfolded protein response to cope with endoplasmic reticular stress. Changes in the mitochondrial membrane potential and generation of superoxide provided further evidence that DENV-2 induces oxidative stress in both C6/36 and BHK-21 cells. Significant elevation of glutathione S-transferase (GST) activity was shown in infected C6/36, but not BHK-21, cells, while suppression of GST produced superoxide at 36 hpi and increased the cell death rate at 48 hpi. This indicates that mosquito cells protect themselves against viral infection through antioxidant defenses.
Assuntos
Antioxidantes/fisiologia , Culicidae/imunologia , Vírus da Dengue/imunologia , Animais , Apoptose , Linhagem Celular , Cricetinae , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Potencial da Membrana Mitocondrial , Estresse Oxidativo , Superóxidos/toxicidade , Resposta a Proteínas não DobradasRESUMO
Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , RNA Viral/genética , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Primers do DNA/genética , Vírus da Encefalite Japonesa (Espécie)/classificação , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Evolução Molecular , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , RNA Viral/química , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transfecção , Regiões não TraduzidasRESUMO
BACKGROUND: Clinical diagnosis of Japanese encephalitis is usually difficult due to non-specific signs at the early and acute stages of the infection. Virus isolation from peripheral blood is also not possible because of the short period and low level of transient viremia even in the acute stage of the disease. It is thus urgent to develop a feasible and convenient method for laboratory diagnosis of the infection. OBJECTIVES: To establish a newly designed molecular approach that can be used to detect intracellular Japanese encephalitis viral RNA in host cells. STUDY DESIGN: The method was firstly established and then was carried out to test its efficacy in cultured BHK-21 cells, subsequently in peripheral blood mononuclear cells (PBMCs) isolated from mice that have been inoculated with JE virus suspension. RESULTS: In this study, in situ reverse-transcription loop-mediated isothermal amplification (in situ RT-LAMP) was established; which combines merits of recently developed loop-mediated isothermal amplification (LAMP) and in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). CONCLUSIONS: The newly designed method can detect viral RNAs in peripheral blood mononuclear cells (PBMCs) in a short time with high sensitivity and efficiency.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Encefalite Japonesa/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Animais , Linhagem Celular , Cricetinae , Vírus da Encefalite Japonesa (Espécie)/genética , Feminino , Camundongos , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Dengue (Den) viruses cause apoptosis in mammalian cells, but usually result in high progeny yields without evident damage in mosquito cells. By using subtractive hybridization, 13 potentially virus-induced genes were selected in Den-2 virus-infected Aedes albopictus C6/36 cells. Based on semi-quantitative and real-time RT-PCR, one novel gene, named C189, was significantly upregulated in infected C6/36 cells. Its full-length of 678 nucleotides (nt) was determined by a combination of 5'- and 3'-RACE products. After alignment, C189 was classified as a member of the tetraspanin superfamily that typically has 2 short cytoplasmic sequences, 4 transmembrane domains, as well as small and large extracellular regions (EC1 and EC2). It contains the hallmark CCG motif in the EC2 region and additional 17 conserved nucleotides as do other tetraspanins. C189 was not upregulated by inoculation of UV-inactivated Den-2 virus to C6/36 cells. This suggests that tetraspanin upregulation is not related to virus binding to the cell surface, and that C189 does not function as a receptor for dengue virus entry. On the other hand, overexpression of C189 was concurrent with viral proteins, targeting the plasma membrane of C6/36 cells infected with Den-2 virus. It is presumably beneficial or essential for cell-to-cell spread of the virus due to the role of tetraspanins demonstrated in intercellular adhesion.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Proteínas de Membrana/biossíntese , Aedes/química , Aedes/genética , Aedes/virologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/química , Sequência Conservada , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Microscopia Confocal , Dados de Sequência Molecular , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Genetically different subpopulations were identified and purified from Japanese Encephalitis virus (JEV). Those with small plaques (SPs; <2 mm in diameter), derived from strains of T1P1, CJN, and CC27, were more competent than those with large plaques (LPs; >5 mm in diameter) when passaged in Neuro-2a cells. Differences in amino acids between SPs and LPs from each strain were shown in the viral envelope (E) protein. The amino acid at E-306 was Glu in LP but was substituted by Lys in SP in the T1P1 strain. A similar substitution occurred at E-138 in the CJN strain. However, the amino acid was Asp in LP but was substituted by Asn in SP at E-389 in the CC27 strain. All SPs were shown to have a higher affinity to the cellular membrane when compared to LPs, and this resulted in more-efficient infection of Neuro-2a cells, suggesting that the differential fitness of JEV variants to Neuro-2a cells appeared in the early phase of infection. In addition, glycosaminoglycans (GAGs) on the surface of many mammalian cells have been demonstrated to be critical for infection by JEV, especially SP variants. The present results suggest that T1P1-SP1 viruses infected Neuro-2a cells more efficiently in spite of the sparse distribution of cell surface GAGs. We conclude that highly sulfated forms of GAGs expressed by Neuro-2a cells play an important role in selecting JEV variants with specific mutations in the E glycoprotein.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Glicosaminoglicanos/metabolismo , Neurônios/virologia , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Cricetinae , Análise Mutacional de DNA , Vírus da Encefalite Japonesa (Espécie)/genética , Heparitina Sulfato/análise , Heparitina Sulfato/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Análise de Sequência de DNA , Proteínas do Envelope Viral/genética , Ensaio de Placa ViralRESUMO
Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2-3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides.