Quantitation of Friend spleen focus-forming virus by a nine-day 59Fe assay.
A previously described 3-day 59Fe assay for quantitation of Friend spleen focus-forming virus has been modified to produce a 200-fold more sensitive 9-day 59Fe assay. A characterization of this assay is reported here. Male BALB/c mice received intravenous injections of appropriately diluted Friend polycythemia virus (FVP); control mice received virus diluent. All mice were allowed food and water ad libitum for 6 days, and on day 6 after virus injection were fasted by removal of food but not water. On day 3 of the fast (the 9th day after virus injection) each mouse received an intraperitoneal injection of 1 muCi of 59Fe. Six hours later the mice were sacrificed and the splenic radioactivity was determined. The percent splenic incorporation of 59Fe was directly related to the logarithm of spleen focus-forming units (SFFU) of FVP injected in a range of approximately 25 to 1,000 SFFU. Using a standard FVP preparation in a dose range of 25 to 1,000 SFFU, it was possible to determine the SFFU titers of unknown samples by extrapolation of the percent splenic 59Fe incorporation to the logarithm of SFFU. SFFU titers obtained by the 9-day 59Fe assay were similar to those obtained by the enumerative-response assay. Advantages of the 9-day 59Fe assay over the enumerative-response assay include a 50-fold greater virus dose range, an easier and a more objective counting procedure, and a reduced coefficient of variation. (+info)
A new picornavirus isolated from bank voles (Clethrionomys glareolus).
A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype. (+info)
Rubella virus-induced apoptosis varies among cell lines and is modulated by Bcl-XL and caspase inhibitors.
Rubella virus (RV) causes multisystem birth defects in the fetuses of infected women. To investigate the cellular basis of this pathology, we examined the cytopathic effect of RV in three permissive cell lines: Vero 76, RK13, and BHK21. Electron microscopy and the TUNEL assay showed that the cytopathic effect resulted from RV-induced programmed cell death (apoptosis) in all three cell lines, but the extent of apoptosis varied among these cells. At 48 h postinfection, the RK13 cell line showed the greatest number of apoptotic cells, the Vero 76 cell line was approximately 3-fold less, and BHK21 had very few. An increased multiplicity of infection and longer time postinfection were required for the BHK21 cell line to reach the level of apoptotic cells in Vero 76 at 48 h. Purified RV induced apoptosis in a dose-dependent fashion, but not UV-inactivated RV or virus-depleted culture supernatant. Specific inhibitors of the apoptosis-specific proteases caspases reduced RV-induced apoptosis and led to higher levels of RV components in infected cells. To address the role of regulatory proteins in RV-induced apoptosis, the antiapoptotic gene Bcl-2 or Bcl-XL was transfected into RK13 cells. Although a high level of Bcl-2 family proteins was expressed, no protection was observed from apoptosis induced by RV, Sindbis virus, or staurosporine in RK13 cells. In BHK21 cells, however, increased expression of Bcl-XL protected cells from apoptosis. The observed variability in apoptotic response to RV of these cell lines demonstrates that programmed cell death is dependent on the unique properties of each cell and may be indicative of how selective organ damage occurs in a congenital rubella syndrome fetus. (+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)
Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes.
Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications. (+info)
Serum albumin inhibits echovirus 7 uncoating.
Echoviruses induce a wide spectrum of diseases in man, the most severe being meningitis. In neonates, however, a severe systemic infection can be observed, leading to death. Serum albumin is the most abundant protein in plasma and most interstitial fluids, and its functions include osmoregulation and transport and delivery of hydrophobic molecules such as fatty acids and steroids. The results of cold-synchronized one-step growth analysis of echovirus 7 infection and sucrose-gradient analysis of A-particles suggest that physiological concentrations of albumin block echovirus 7 infection by inhibiting uncoating. The blockage was reversible and was still effective when albumin was added 30 min after virus adsorption. Inhibition of uncoating was confirmed by using rhodanine, a known specific inhibitor of echovirus uncoating. After removal of the albumin blockage, addition of rhodanine perpetuated the inhibition. Serum and interstitial albumin concentrations may limit echovirus infection in vivo and thereby act as an extracellular determinant for echovirus tropism. (+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)
Activation of caspases and p53 by bovine herpesvirus 1 infection results in programmed cell death and efficient virus release.
Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the G0/G1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and P.-P. Pastoret, Virology 232:351-358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638-7641, 1998). The mechanism by which BHV-1 induces PCD in peripheral blood mononuclear cells is not understood, nor is it clear whether nonlymphoid cells undergo PCD following infection. This study demonstrates that infection of bovine kidney (MDBK) cells with BHV-1 leads to PCD, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, DNA laddering, and chromatin condensation. p53 appears to be important in this process, because p53 levels and promoter activity increased after infection. Expression of proteins that are stimulated by p53 (p21(Waf1) and Bax) is also activated after infection. Cleavage of Bcl-xL, a protein that inhibits PCD, occurred after infection, suggesting that caspases (interleukin-1beta-converting enzyme-like proteases) were activated. Other caspase substrates [poly(ADP-ribose) polymerase and actin] are also cleaved during the late stages of infection. Inhibition of caspase activity delayed cytotoxic activity and virus release but increased the overall virus yield. Taken together, these results indicate that nonlymphoid cells undergo PCD near the end of productive infection and further suggest that caspases enhance virus release. (+info)