Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

Renibacterium salmoninarum is a Gram-positive bacterium causing bacterial kidney disease (BKD) in susceptible salmonid fishes. Several quantitative PCR (qPCR) assays to measure R. salmoninarum infection intensity have been reported, but comparison and evaluation of these assays has been limited. Here, we compared 3 qPCR primer/probe sets for detection of R. salmoninarum in field samples of naturally exposed Chinook and coho salmon first identified as positive by nested PCR (nPCR). Additional samples from a hatchery population of Chinook salmon with BKD were included to serve as strong positive controls. The 3 qPCR assays targeted either the multiple copy major soluble antigen (msa) genes or the single copy abc gene. The msa/non-fluorescent quencher (NFQ) assay amplified R. salmoninarum DNA in 53.2% of the nPCR positive samples, whereas the abc/NFQ assay amplified 21.8% of the samples and the abc/TAMRA assay 18.2%. The enzyme-linked immunosorbent assay (ELISA) successfully quantified only 16.4% of the nPCR positive samples. Although the msa/NFQ assay amplified a greater proportion of nPCR positive samples, the abc/NFQ assay better amplified those samples with medium and high ELISA values. A comparison of the geometric mean quantity ratios highlighted limitations of the assays, and the abc/NFQ assay strongly amplified some samples that were negative in other tests, in contrast to its performance among the sample group as a whole. These data demonstrate that both the msa/NFQ and abc/NFQ qPCR assays are specific and effective at higher infection levels and outperform the ELISA. However, most pathogen studies will continue to require multiple assays to both detect and quantify R. salmoninarum infection.     

An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.     

Antiviral function of tilapia hepcidin 1–5 and its modulation of immune-related gene expressions against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells

Antimicrobial peptides, small cysteine-rich molecules, play vital roles in host defense mechanisms against pathogen infection. Recently, tilapia hepcidin (TH)1–5, was characterized, and its antimicrobial functions against several pathogens were reported. Herein, we investigated the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The presence of TH1-5 enhanced the survival of CHSE-214 cells infected with IPNV. Additionally, the number of plaques formed by the cytopathic effect of IPNV in CHSE-214 cells decreased when IPNV was preincubated with TH1-5. This observation demonstrates the antiviral function of TH1-5. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. When TH1-5 and IPNV were used to co-treat CHSE-214 cells, then cells were re-challenged with IPNV at 24 h, the cells did not survive the IPNV infection. This shows that in the absence of TH1-5, viral re-challenge killed CHSE-214 cells. In conclusion TH1-5 protected CHSE-214 cells against IPNV by direct antimicrobial and immunomodulatory functions.     

An Alternative Efficient Procedure for Purification of the Obligate Intracellular Fish Bacterial Pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.     

Sequencing, cloning and expression of a β-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4

Abstract We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is qonserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.     

Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.Abbreviations BSA bovine serum albumin - CHSE-214 Chinook samon embryo cell line - dFBS dialyzed fetal bovine serum - FBS fetal bovine serum - GFSk-S1 goldfish skin cell line - GS glutamine synthetase - L-15 Leibovitz's L-15 media - L929 mouse fibroblast cell line - MEM minimum essential medium Eagle - PBS phosphate buffered saline - RTL-W1 rainbow trout liver cell line - RTSp-W1 rainbow trout spleen cell line     

Characterization of the sortase repertoire in Bacillus anthracis

LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss), bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins.     

Effect of purine supplementation on the growth of salmonid cell lines in different mammalian sera

The Chinook salmon embryo cell line, CHSE-214, grew well in fetal bovine serum (FBS) but poorly in dialyzed (d) FBS. Purines restored most but not all growth-promoting activity to dFBS, which suggests that purines account for a large portion of the dialyzable fraction's growth-promoting activity. CHSE-214 died in newborn calf serum (NCS) but grew slightly in dNCS, which suggests that the dialyzable fraction of NCS contains a toxic component(s). Little or no proliferation occurred in calf serum (CS); some took place in horse serum (HS). Porcine serum (PS) was very toxic. In all these sera except PS and HS, the purine nucleoside, inosine, significantly enhanced growth, whereas the pyrimidine nucleoside, uridine, was without effect. The other purines, hypoxanthine, adenine, adenosine and guanosine also stimulated proliferation but not as well as inosine. Inosine also enhanced the growth of the rainbow trout gonadal cell line, RTG-2. Although their morphology underwent minor alterations in medium with inosine, CHSE-214 cells could be grown indefinitely in CS and inosine as effectively as in the more expensive FBS.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Gliotoxin-induced cytotoxicity in three salmonid cell lines: cell death by apoptosis and necrosis

Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 microM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 microM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation.     

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.     

A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum

Abstract By means of a one-step one-tube extraction from less than 1 mg of tissue it is possible to identify, via the polymerase chain reaction, Renibacterium salmoninarum in salmon with bacterial kidney disease. A 149-bp DNA sequence unique to R salmoninarum was specifically amplified and its nature confirmed by Southern hybridization using a non-isotopically labelled probe. The sensitivity of the approach allowed the detection of 22 R. salmoninarum cells. The procedure was successfully applied in the identification of the causative agent of bacterial kidney disease in kidney tissue from infected fishes.     

Detection of Renibacterium salmoninarum antigen in migrating adult chum salmon (Oncorhynchus keta) in Japan.

Renibacterium salmoninarum antigen was detected in the kidney of migrating chum salmon (Oncorhynchus keta) using the indirect dot blot assay and indirect fluorescent antibody test. The adult chum salmon had migrated into a bay in which cultured coho salmon infected with R. salmoninarum were present. Antigen was detected in 5% of the chum salmon although they did not have clinical signs of bacterial kidney disease (BKD). This report describes the first case of R. salmoninarum antigen detection among wild chum salmon populations in eastern Asia.     

Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha

Laboratory studies of the transmission and pathogenesis of Renibacterium salmoninarum may describe more accurately what is occurring in the natural environment if test fish are infected by waterborne R. salmoninarum shed from infected fish. To quantify bacterial shedding by chinook salmon Oncorhynchus tschawytscha at 13 degrees C in freshwater, groups of fish were injected intraperitoneally with R. salmoninarum at either 1.3 x 10(6) colony forming units (CFU) fish (-1) (high-dose injection group) or 1.5 x 10(3) CFU fish (-1) (low-dose injection group). R. salmoninarum infection levels were measured in the exposed fish by the enzyme-linked immunosorbent assay (BKD-ELISA). At regular intervals for 30 d, the numbers of R. salmoninarum shed by the injected fish were calculated on the basis of testing water samples by the membrane filtration-fluorescent antibody test (MF-FAT) and bacteriological culture. Mean BKD-ELISA optical densities (ODs) for fish in the low-dose injection group were not different from those control fish (p > 0.05), and no R. salmoninarum were detected in water samples taken up to 30 d after injection of fish in the low-dose group. By 12 d after injection a proportion of the fish from the high-dose infection group had high (BKD-ELISA OD > or = 1.000) to severe (BKD-ELISA OD > or = 2.000) R. salmoninarum infection levels, and bacteria were detected in the water by both tests. However, measurable levels of R. salmoninarum were not consistently detected in the water until a proportion of the fish maintained high to severe infection levels for an additional 8 d. The concentrations of R. salmoninarum in the water samples ranged from undetectable up to 994 cells ml(-1) on the basis of the MF-FAT, and up to 1850 CFU ml(-1) on the basis of bacteriological culture. The results suggest that chinook salmon infected with R. salmoninarum by injection of approximately 1 x 10(6) CFU fish (-1) can be used as the source of infection in cohabitation challenges beginning 20 d after injection.     

Prevalence and analysis of Renibacterium salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha in North Puget Sound

Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks.     

Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli

Type I (alpha/beta) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-alpha (SasaIFN-alpha1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-alpha1 (rSasaIFN-alpha1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-alpha subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-alpha, the mature form of the SasaIFN-alpha2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-alpha2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-alpha2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 x 10(6) U mg(-1) against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-alpha2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-alpha2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.     

Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA

The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.