Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.     

DNA Immunization with Japanese Encephalitis Virus Nonstructural Protein NS1 Elicits Protective Immunity in Mice

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1′), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1′ could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1′ coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.     

Recognition of Nonstructural Protein Peptides by Cytotoxic T Lymphocytes Raised against Japanese Encephalitis Virus

We have previously reported that Lyt2⁺ cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K^d-restricted CTL and to examine their cross-recognition of West Nile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K^d binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.     

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues ⁵AIDITRK¹¹, ⁷²RDELNVL⁷⁸, ²⁵¹KSKHNRREGY²⁶⁰, ²⁶⁹DENGIVLD²⁷⁶, and ³⁴¹DETTLVRS³⁴⁸. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.     

Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.     

单克隆抗体治疗日本脑炎病毒感染恒河猴的实验研究

感染日本脑炎病毒(JEV)的恒河猴,出现脑炎的一系列症状与体征,终因呼吸及循环衰竭而死亡。JEV感染猴体温升到超过39℃以上后的第2天,注射单克隆抗体(McAb)制剂进行实验性治疗。结果显示,McAb经肌肉、静脉、硬脑膜下加肌肉等不同途径注射后,均有良好的疗效,对恒河猴的保护率达100％,其中以硬膜下加肌肉途径注射McAb的效果最佳。多次注射McAb,未发现任何不良反应与毒副作用。本研究表明,将鼠源性McAb用于灵长类安全可靠,若过渡到临床使用,是可行的。     

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.     

Proliferative Response of Human Peripheral Blood Mononuclear Cells to Japanese Encephalitis Virus

Cell-mediated immune response to Japanese encephalitis virus (JEV) and its purified envelope (E) protein was measured in 45 laboratory confirmed JE patients using a proliferation assay of peripheral blood mononuclear cells (PBMC). In parallel, JEV-specific IgM antibodies were measured by ELISA. No correlation was observed between the antibody response and results of the lymphocyte proliferation assay. Only 11 of the 42 patients positive in the antibody test were positive in the proliferation assay, and PBMC from 14/45 (31%) patients did not respond to either phytohemagglutinin and to JEV and/or its purified E protein antigen. No correlation was observed between the cell-mediated immune response and the final clinical outcome (fatality vs. recovery).     

Virus-Specific Proteins Synthesized in Cells Infected with RNA+ Temperature-Sensitive Mutants of Sindbis Virus

All Sindbis virus temperature-sensitive mutants defective in "late" functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that (3)H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive.     

乙脑病毒持续感染株preM区序列分析

为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒.利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较.preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸).对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同.认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关.     

乙脑病毒持续感染株preM区序列分析

为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒。利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较。preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸)。对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同。认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关。     

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. This genus is divided into the Old World and New World alphaviruses, which demonstrate profound differences in pathogenesis, replication, and virus-host interactions. VEEV is a representative member of the New World alphaviruses. The biology of this virus is still insufficiently understood, particularly the function of its nonstructural proteins in RNA replication and modification of the intracellular environment. One of these nonstructural proteins, nsP3, contains a hypervariable domain (HVD), which demonstrates very low overall similarity between different alphaviruses, suggesting the possibility of its function in virus adaptation to different hosts and vectors. The results of our study demonstrate the following. (i) Phosphorylation of the VEEV nsP3-specific HVD does not play a critical role in virus replication in cells of vertebrate origin but is important for virus replication in mosquito cells. (ii) The VEEV HVD is not required for viral RNA replication in the highly permissive BHK-21 cell line. In fact, it can be either completely deleted or replaced by a heterologous protein sequence. These variants require only one or two additional adaptive mutations in nsP3 and/or nsP2 proteins to achieve an efficiently replicating phenotype. (iii) However, the carboxy-terminal repeat in the VEEV HVD is indispensable for VEEV replication in the cell lines other than BHK-21 and plays a critical role in formation of VEEV-specific cytoplasmic protein complexes. Natural VEEV variants retain at least one of the repeated elements in their nsP3 HVDs.     

Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.     

NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase–NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of ~50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.     

流行性乙型脑炎病毒在Vero细胞上传代适应的研究

通过乙脑病毒Ｐ＿３株以不同方式在鼠脑和Ｖｃｒｏ细胞间传代适应的研究,培育出适于工业化大生产制备乙脑疫苗的生产毒种。其毒力可达７．０±０．５ｌｇＬＤ５０／０．０４ｍｌ,空斑滴度为７．５±０．５ＰＦＵ／ｍｌ,免疫原性ＩＤ５０值为０．００００１６－０．００００２３ｍｌ;在含有适量人白蛋白的１９９营养液内,该毒种于－３０℃至少可稳定一年;与鼠脑组织毒种相比,传代细胞毒种的毒力、免疫原性及稳定性均与其相当。然而,后者不含脑组织,至少降低了一种过敏的危险。另外,细胞毒种的制备无需手工解剖鼠脑,易于控制外源因子污染,易于标准化。对于大规模生产疫苗、提高疫苗产量和质量,传代细胞毒种比鼠脑组织悬液毒种具有明显的优点。     

Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010

After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30–39, 40–49, 50–59, and 60–69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (2.2%) subjects were found to have been recently infected by the virus. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea.     

Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC₅₀ = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Virus-Specific Regulatory T Cells Ameliorate Encephalitis by Repressing Effector T Cell Functions from Priming to Effector Stages

Several studies have demonstrated the presence of pathogen-specific Foxp3⁺ CD4 regulatory T cells (Treg) in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg) and conventional CD4 T cells (M133 Tconv) in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN). Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.     

乙型脑炎病毒可视化分型基因芯片的制备

目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。