Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.
The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s). (+info)
Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.
The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses. (+info)
Induction of CD8+ T cell-mediated protective immunity against Trypanosoma cruzi.
Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circum-sporozoite (CS) protein, which contains the well-characterized CD8+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection. (+info)
Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.
The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine. (+info)
Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada.
Infectious salmon anemia virus (ISAV) was isolated at a marine grow-out site in New Brunswick, Canada, from Atlantic salmon Salmo salar which experienced mortalities due to hemorrhagic kidney syndrome (HKS). Of 20 fish sampled in this study, 14 showed histologically various degrees of interstitial hemorrhaging, tubular epithelial degeneration and necrosis, and tubular casts in the posterior kidney, typical of HKS. Posterior kidney and spleen homogenates produced a cytopathic effect on chinook salmon embryo (CHSE-214) cells 10 to 14 d after inoculation. Pleomorphic virus particles in the size range 80 to 120 nm were seen by electron microscopy. The virus was confirmed as ISAV using reverse transcriptase-polymerase chain reaction (RT-PCR). This is a systematic diagnostic study of the isolation of ISAV on the North American continent and the first description of the growth of ISAV on the CHSE-214 cell line. (+info)
Genomic relationships of the North American isolate of infectious salmon anemia virus (ISAV) to the Norwegian strain of ISAV.
Nucleotide and deduced amino acid sequences were determined for a 436 bp reverse transcriptase-polymerase chain reaction (RT-PCR) cDNA fragment from genome segment 8 and a 1151 bp RT-PCR cDNA fragment from genome segment 2 of the North American isolate of infectious salmon anemia virus (ISAV) and compared to the published sequences of Norwegian isolates of ISAV. The North American ISAV isolate exhibited 82.9% identity with the Sotra 92/93 ISAV isolate from Norway in the partial cDNA sequence of genome segment 2, which encodes a polymerase component protein (PB1). The North American ISAV exhibited 88 and 89% identity with 2 partial cDNA sequences of genome segment 8 (nonstructural, NS, gene) reported for the Glesvaer/2/90 isolate from Norway. The North American ISAV exhibited 96.6% similarity with the Sotra 92/93 ISAV isolate from Norway in the deduced amino acid sequences of the PB1 protein. The deduced amino acid sequence of the protein encoded in the partial cDNA fragment of open reading frame (ORF) 1 of genome segment 8 of the North American ISAV exhibited only 71.2 and 66.7% similarity with the 2 sequences of the Norwegian Glesvaer/2/90 isolate. However, the North American ISAV isolate exhibited 96.2 and 87.2% similarity with the 2 sequences of the Norwegian Glesvaer/2/90 isolate in the deduced amino acid sequences of the protein encoded in the partial cDNA of ORF 2. Comparison of these partial cDNA nucleotide and deduced amino acid sequences confirmed that the North American isolate is ISAV. However, the differences observed in these genomic sequences suggest that the North American isolate may represent a distinct genomic variant from the previously described Norwegian strains. (+info)
First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome.
Haemorrhagic kidney syndrome (HKS), a serious disease affecting Atlantic salmon on the east coast of Canada, was determined to be caused by infectious salmon anaemia virus (ISAV) through the isolation of the pathogen on the SHK-1 (salmon head kidney) cell line and confirmation by ISAV-specific immunofluorescent antibody test (IFAT) and reverse transcriptase polymerase chain reaction (RT-PCR). In addition, the defining histopathology of HKS could be reproduced following the injection of material that rendered challenged fish ISAV-positive by cell culture in the absence of any other detectable pathogen. Preliminary nucleotide sequence comparison does not suggest any direct epidemiological connection between the Canadian and Norwegian isolates. (+info)
Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza A viruses.
Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza. A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6. The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 degrees C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals. (+info)