Analysis of microRNAs induced by Venezuelan equine encephalitis virus infection in mouse brain

MicroRNAs (miRNA) are small RNA (∼22nts) molecules that are expressed endogenously in cells and play an important role in regulating gene expression. Recent studies have shown that cellular miRNA plays a very important role in the pathogenesis of viral infection. Venezuelan equine encephalitis virus (VEEV) is an RNA virus and is a member of the genus Alphavirus in the family Togaviridae. VEEV is infectious in aerosol form and is a potential biothreat agent. In this study, we report for the first time that VEEV infection in mice brain causes modulation of miRNA expression. Pathway analyses showed that majority of these miRNAs are involved in the neuronal development and function. Target gene prediction of the modulated miRNAs correlates with our recently reported mRNA expression in VEEV infected mice brain.     

Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein

A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity (approximately 7nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioaptamer in a native gel.     

Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

Analysis of the survival of Venezuelan equine encephalomyelitis virus and possible viral simulants in liquid suspensions

Aims: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. Methods and Results: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4°C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30°C were very similar (between 0·12 and 0·14 log₁₀ per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21°C, VEE and SV were inactivated at twice the rate measured in dd water. Conclusions: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. Significance and Impact of the Study: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE.     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

东部马脑脊髓炎病毒的分子生物学进展

东部马脑脊髓炎病毒属虫媒病毒,能引起人和马发生急性脑炎。东马病毒为单股正链RNA病毒,可分为南美型和北美型,包括两个开放读码框架,分别编码结构蛋白（E1,E2,E2,C,6K）和非结构蛋白（nsp1,nsp2,nsp3,nsp4)。其中E1/E2包膜糖蛋白以异二聚体的形式病毒颗粒外刺突。非结构蛋白主要能与负链RNA的合成,近来,随着研究深入,病毒受体越来越受到广泛关注。本介绍东部马脑脊髓炎病毒结构、进化、复制、组装等方面的分子生物学进展。     

Human-like antibodies neutralizing Western equine encephalitis virus

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x10⁴ TCID₅₀/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development.     

Human-like antibodies neutralizing Western equine encephalitis virus

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x10⁴ TCID₅₀/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development.     

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463–785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.     

Cellular laminin-binding protein and Venezuelan equine encephalomyelitis virus replication in Vero, 293 and 9S2 cells

The expression of the laminin-binding protein (LBP) on cellular membranes in different cell lines has been studied. A high level of replication of Venezuelan equine encephalomyelitis (VEE) virus was registered in Vero cells with high levels of LBP on the cell surface. The treatment of Vero cells with monoclonal antibodies to human LBP reduced VEE virus replication by a factor of more than 200. A low level of LBP expression on the surface of 293 cells was increased via transfection by plasmid with gene for human LBP. The VEE virus replication in transfected cells (9S2) was increased by more that 2000 times compared to the 293 cells. The results demonstrated the principal role of cellular LBP in the entry of VEE virus into mammalian cells. It is proposed that LBP is a key cellular protein for the early stage of the VEE virus replication in cells. LBP may be a target protein for the development of a new generation of antiviral drugs capable of inhibiting (enhancing) the alphavirus replication in human cells.     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立

目的:表达委内瑞拉马脑炎病毒E2重组蛋白,并结合胶体金免疫层析技术建立一种简便检测委内瑞拉马脑炎病毒特异性抗体的方法。方法:利用已经构建的表达E2抗原的工程菌, 用IPTG诱导, 表达蛋白主要以包涵体的形式存在。通过一系列条件的变性、复性、透析,所制抗原用以包被硝酸纤维素膜,利用胶体金标记和免疫层析技术,建立委内瑞拉马脑炎病毒快速检测方法。对该方法的敏感性、特异性和稳定性作出评价。结果:重组工程菌可表达分子质量为 40 kDa的目的蛋白, 纯化后的蛋白质经SDS-PAGE显示纯度达95%以上。建立的检测方法可在20 min内完成检测。对症状相似及近缘的其他病毒进行检测,均无非特异反应。试纸条在37℃下保存2周,检测结果不变。该方法与R&;D公司商品化的ELISA试剂盒灵敏度检测无明显差异;对92份阴性血清进行检测,两种检测方法的符合率为96.7%。结论:重组委内瑞拉马脑炎病毒蛋白产生的包涵体变性复性后生具有良好的重复性和稳定性, 可作为委内瑞拉马脑炎病毒多种检测方法的抗原原料。胶体金免疫层析法具有快速、灵敏、特异、稳定的特点,适用于现场检测。     

Antagonistic Effect of Luzindole in Mice Treated with Melatonin During the Infection with the Venezuelan Equine Encephalomyelitis Virus

The effect of Luzindole (LZ) in mice treated with melatonin (MEL) during the infection with the Venezuelan equine encephalomyelitis (VEE) virus was examined. Melatonin (500 μg/Kg b.w.) was administered daily 3 days before and 5 days after the infection. Luzindole (5 mg/Kg b.w.) was injected intraperitoneally 3 days before (pre-infection) or 5 days after (post-infection) the infection. Mortality rates in the infected mice treated both with MEL and LZ were higher than in those treated with MEL alone in which the lowest brain and serum viral titers were detected. On the third post-infection day, viral titers of the MEL + VEE + LZ (pre-infection) group were higher than those of the remainder groups. On the fifth day, viral titers in infected mice were similar to those of the MEL + VEE + LZ (pre-infection) group, but higher than those detected in the MEL + VEE + LZ (post-infection). In conclusion, the protective effect of MEL in mice infected with VEE virus was inhibited by LZ suggesting that this protection is mediated by MEL receptors.     

Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis

The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which migrate to the draining lymph node following activation. A point mutation in the E2 glycoprotein gene of VEE that renders the virus avirulent and compromises its ability to spread beyond the draining lymph blocked the appearance of virally infected dendritic cells in the lymph node in vivo. A second-site suppressor mutation that restores viral spread to lymphoid tissues and partially restore virulence likewise restored the ability of VEE to infect dendritic cells in vivo.     

Liquid-phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus.

Ozone, in a liquid-phase application, was evaluated as a residue-free viral inactivant that may be suitable for use in an arboviral research laboratory. Commonly used sterilizing agents may leave trace residues, be flammable or explosive, and require lengthy periods for gases or residues to dissipate after decontamination of equipment such as biological safety cabinets. Complete liquid-phase inactivation of Venezuelan equine encephalomyelitis virus was attained at 0.025 mg of ozone per liter within 45 min of exposure. The inactivation of 10(6.5) median cell culture infective doses (CCID50 of Venezuelan equine encephalomyelitis virus per milliliter represented a reduction of 99.99997% of the viral particles from the control levels of 10(7.25-7.5) CCID50/ml. A dose-response relationship was demonstrated. Analysis by polynomial regression of the logarithmic values for both ozone concentrations and percent reduction of viral titers had a highly significant r2 of 0.8 (F = 63.6; df = 1, 16). These results, together with those of Akey (J. Econ. Entomol. 75:387-392, 1982) on the use of ozone to kill a winged arboviral vector, indicate that ozone is a promising candidate as a sterilizing agent in some applications for biological safety cabinets and other equipment used in vector studies with arboviruses.