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Twelve lytic viruses (PgV) infecting the marine unicellular eukaryotic harmful algal bloom species Phaeocystis globosa were isolated from the southern North Sea in 2000–2001 and partially characterized. All PgV isolates shared common phenotypic features with other algal viruses belonging to the family Phycodnaviridae and could be categorized in four different groups. Two main groups (PgV Group I and...
Current smallpox vaccines are live vaccinia viruses that replicate in the vaccinee inducing immunity against the deadly disease smallpox. Replication resulting in virus spread within the host, however, is the major cause of severe postvaccinal adverse events. Therefore, attenuated strains such as modified vaccinia Ankara (MVA) or LC16m8 are candidates as next generation vaccines. These strains are...
We generated a high-growth H5N1/PR8 virus by plasmid-based reverse genetics. The virulence associated multiple basic amino acids of the HA gene were removed, and the resulting virus is attenuated for chickens and chicken eggs. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When SPF chickens were inoculated with 0.3 ml of the vaccine, the hemagglutinin-inhibition (HI) antibody...
We reported that immunization with recombinant proteins derived from vaccinia virus (VV) particles could provide protection against infection. Here we describe the physical and antigenic properties of the L1R membrane protein. The recombinant protein (L1R(185t)) was secreted as a monomer and correct folding was suggested by the presence of three intramolecular disulfide bonds and binding to conformation-specific...
Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically...
Here, we report the diversity of amantadine-resistant mutants among avian influenza A viruses with pandemic potential (H5, H6, H7, and H9 hemagglutinin subtypes). Drug-resistant variants were not detected among 1979–83 isolates, whereas 31.1% of H5 and 10.6% of H9 strains from Southeast Asia isolated in 2000–04 carried mutations in M2 protein. In North America, resistant variants occurred among H7...
We generated recombinant A/WSN/33 influenza A viruses expressing a PB2 protein fused to a Flag epitope at the N- (Flag-PB2) or C-terminus (PB2-Flag), which replicated efficiently and proved to be stable upon serial passage in vitro on MDCK cells. Rescue of PB2-Flag viruses required that the 5′ end of the PB2 segment was kept identical to the wild-type beyond the 34 noncoding terminal nucleotides....
The order of genes in the genome is commonly thought to have functional significance for gene regulation and fitness but has not heretofore been tested experimentally. We adapted a bacteriophage T7 variant harboring an ectopically positioned RNA polymerase gene to determine whether it could regain the fitness of the wild type. Two replicate lines maintained the starting gene order and showed only...
The fusion subunit of the SARS-CoV S glycoprotein contains two regions of hydrophobic heptad-repeat amino acid sequences that have been shown in biophysical studies to form a six-helix bundle structure typical of the fusion-active core found in Class I viral fusion proteins. Here, we have applied serine-scanning mutagenesis to the C-terminal-most heptad-repeat region in the SARS-CoV S glycoprotein...
The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous...
We describe here an in vitro system for template-dependent initiation and synthesis of a Sindbis virus (SV) subgenomic (SG) RNA transcript. The critical components of this system were (1) a minus-strand promoter-template corresponding to the region of the SV genome from nt 7441 to nt 7772 (−157 to +175 relative to the SG RNA transcription initiation site at nt 7598), and (2) a p15 fraction from cells...
Entry of HIV and SIV into susceptible cells is mediated by CD4 and chemokine receptors, which act as coreceptors. To study cell entry of SIV, we constructed a cell line, xKLuSIV, derived from non-susceptible human K562 cells, that express the firefly luciferase reporter gene under control of a minimal SIV long terminal repeat (LTR). Using these susceptible cells, we studied the entry of a well-characterized...
Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-α and...
Ineffective transgene expression in a sufficient amount of target cells is still a limitation in retroviral vector mediated gene therapy. Thus, we systematically evaluated four genetic modulators, (i) the woodchuck posttranscriptional regulatory element (WPRE), (ii) the mouse RNA transport element (RTE), (iii) the constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), and (iv)...
The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we...
Plant viruses, in particular Tobacco mosaic virus (TMV), are model systems to study RNA and protein trafficking in plants. Although TMV cell-to-cell transport controlled by the 30-kDa movement protein (MP) has been intensively studied, it was only recently demonstrated that the 126/183-kDa replicase proteins are also involved in cell-to-cell movement. Elucidating the role(s) of 126/183-kDa proteins...
Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that is packaged into virions and regulates optimal expression of ICP0 and a subset of late (γ) proteins, including UL41 in infected cells. In the present study, we investigated the role(s) of the protein kinase activity of UL13 in viral replication using a recombinant virus expressing enzymatically inactive UL13 after an amino acid substitution...
There is strong interest in developing practical strategies for gene delivery to the central nervous system (CNS). Direct delivery into the brain or spinal cord is highly invasive as well as inefficient or hazardous using most current vector systems. Our objective was to generate innocuous gene vehicles that would be effectively taken up by axons and then home to the neuron cell bodies. Vectors derived...
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