Incorporation of quantum dots on virus in polycationic solution

Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biological imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both negatively charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examining the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 hours, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.     

Glycopeptides from brain inhibit rates of polypeptide chain elongation

In previous reports, we have identified cell-surface glycopeptides from mouse cerebrum (BCSG) that inhibited protein synthesis and mitosis in several cell types. When baby hamster kidney (BHK)-21 cells were infected with vesicular stomatitis virus (a negative strand RNA virus), BCSG extensively inhibited viral protein synthesis. This inhibition was effective against both protein and glycoprotein synthesis and was independent of amino acid uptake by infected cells, synthesis of viral RNA, and degradation of viral proteins. Analysis of polyribosome profiles in uninfected BHK-21 cells indicated that the degree of cellular protein synthesis inhibition could not be attributed to activation of RNase or solely to a disruption of chain initiation. When added directly to a cell-free protein-synthesizing system derived from BHK-21 cells, BCSG was ineffective, but if the inhibitory material was first allowed to react with cells, cell-free protein synthesis was substantially reduced. When BCSG were reacted with cells for 5 min at 0 degrees C, the cells tested, BHK-21 (a BCSG-sensitive line) and murine fibrosarcoma 2237 (a BCSG-insensitive line), both effectively adsorbed the inhibitor from the medium.     

A modified PAP (peroxidase-anti-peroxidase) staining technique using sera from patients with dengue hemorrhagic fever (DHF): 4 step PAP staining technique.

BHK-21 cells infected with dengue virus type 1 were stained by a newly developed 4 step PAP (peroxidase-anti-peroxidase) technique using sera from patients with dengue hemorrhagic fever as anti-virus antibody. The intensity of staining of the sera was proportional to the hemagglutination inhibition and neutralization titers. With this new technique using sera from patients it should be possible to use the PAP technique of virus infections.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.     

Inhibition of dengue virus entry and multiplication into monocytes using RNA interference

Background

Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes.

Methodology/Principal Findings

Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes.

Conclusions/Significance

Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.     

NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition

Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan

ABSTRACT: Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.     

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.     

The antiapoptotic protein Mcl-1 controls the type of cell death in Theiler's virus-infected BHK-21 cells

Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.     

Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway

Although induction of apoptosis by bovine ephemeral fever virus (BEFV) in several cell lines has been previously demonstrated by our laboratory, less information is available on the process of BEFV-induced apoptosis in terms of cellular pathways and specific proteins involved. In order to determine the step in viral life cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection. Treatment of BHK-21 infected cells with ammonium chloride (NH₄Cl) or cells infected with UV-inactivated BEFV was seen to abrogate virus apoptosis induction, suggesting that virus uncoating and gene expression are required for the induction of apoptosis. Using soluble death receptors Fc:Fas chimera to block Fas signaling, BEFV-induced apoptosis was inhibited in cells. BEFV infection of BHK-21 cells results in the Fas-dependent activation of caspase 8 and cleavage of Bid. This initiated the dissipation of the membrane potential and the release of cytochrome c but not AIF or Smac/DIABLO from mitochondrial into cytoplasm leading to activation of caspase 9. Combined activation of the death receptor and mitochondrial pathways results in activation of the downstream effecter caspase 3 leading to cleavage of PARP. Fas-mediated BEFV-induced apoptosis could be suppressed by the overexpression of Bcl-2 or by treatment with caspase inhibitors and soluble death receptors Fc:Fas chimera. Taken together, this study provided first evidence demonstrating that BEFV-induced apoptosis requires viral gene expression and occurs through the activation of Fas and mitochondrion-mediated caspase-dependent pathways. An erratum to this article can be found at     

Molecular Mechanisms Involved in Antibody‐Dependent Enhancement of Dengue Virus Infection in Humans

Dengue is the most common arthropod‐borne viral infection in humans with ～50 million cases annually worldwide. In recent decades, a steady increase in the number of severe dengue cases has been seen. Severe dengue disease is most often observed in individuals that have pre‐existing immunity against heterotypic dengue subtypes and in infants with low levels of maternal dengue antibodies. The generally accepted hypothesis explaining the immunopathogenesis of severe dengue is called antibody‐dependent enhancement of dengue infection. Here, circulating antibodies bind to the newly infecting virus but do not neutralize infection. Rather, these antibodies increase the infected cell mass and virus production. Additionally, antiviral responses are diminished allowing massive virus particle production early in infection. The large infected cell mass and the high viral load are prelude for severe disease development. In this review, we discuss what is known about the trafficking of dengue virus in its human host cells, and the signalling pathways activated after virus detection, both in the absence and presence of antibodies against the virus. This review summarizes work that aims to better understand the complex immunopathogenesis of severe dengue disease.      

The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection

Using flow cytometric assay and monoclonal anti-dengue Ab, we observed that both anti-E and anti-prM Abs could enhance dengue virus infection in a concentration-dependent but serotype-independent manner. Increases were found in both the percentage of dengue-infected cells and the expression of dengue E and NS1 protein per cell. Dengue virion binding and infection were enhanced on FcR-bearing cells via the Fc-FcgammaRII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by the mechanism of peptide (CPFLKQNEPEDIDCW)-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-Ags such as heat shock protein 60. In summary, a novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells.     

Novel antiviral activity found in the media of Sindbis virus-persistently infected mosquito (Aedes albopictus) cell cultures.

Aedes albopictus (mosquito) cells persistently infected with Sindbis virus for a period of 6 months release into the medium a low-molecular-weight material capable of specifically reducing the yields of Sindbis virus during the "acute phase" of infection in mosquito cells. The antiviral activity was produced in detectable levels at 3 days after infection, and its concentration in the extracellular medium increased thereafter. The antiviral activity was inactivated by treatment with the enzyme protease K and heat. It was not activated by treatment with antibody prepared against extracts of Sindbis virus-infected BHK-21 cells. The antiviral activity differs from interferon produced by vertebrate cells in that it is virus specific as well as cell specific.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

登革病毒2型全长cDNA感染性转录体的构建和鉴定

为构建登革病毒感染性克隆, 针对登革病毒2型基因组全长cDNA的体外转录方法及感染性转录体进行研究。采用长链RT-PCR技术, 扩增DEN2 NGC株全长基因组cDNA, 以之为模板, 用SP6 RNA聚合酶系统制备体外转录RNA转录体, 分别经乳鼠脑内接种及电穿孔转染BHK-21细胞, 观察其感染效应。并从受染鼠脑和病变细胞中提取总RNA, 进行RT-PCR扩增、克隆测序以及电镜观察。结果发现, 从感染鼠脑和细胞中经RT-PCR均可扩增出病毒特异的基因片段, 大小与预期一致; 并从乳鼠脑组织和BHK-21细胞中观察到恢复病毒颗粒。上述结果表明本文成功构建的DEN2 NGC株病毒全长cDNA的体外转录体具有感染性, 乳鼠脑内接种途径与电穿孔转染细胞一样可成为体外转录体感染宿主细胞、获得恢复病毒的方法。     

Micromorphological Aspects of the Development of Rubella Virus in BHK-21 Cells

Sequential effects of rubella virus infection in BHK-21 cells were studied by electron microscopy of thin sections of control and infected cells, 2 to 7 days after infection. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of virions from vacuole membranes and involvement of the endoplasmic reticulum. Newly formed endoplasmic reticulum cisternae encircled and segregated virionforming vacuoles together with other cellular elements. Large vacuolar complexes with numerous virus particles developed, and virus release from these areas occurred with disruption at the cell periphery. The viral particles, with a mean diameter of about 56 nm, consisted of an electron-dense core surrounded by a less dense capsid, enveloped by a typical unit membrane derived from the vacuole membrane.     

Neuroblastoma Cell Membranes: Specificity for Cell Fusion Mediated by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Abstract: Temperature-sensitive mutant G3 1 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nucleiin polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.