Identification of gene biomarkers for respiratory syncytial virus infection in a bronchial epithelial cell line

Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to Affymetrix HU133 plus 2.0 microarrays (n = 4). At 4 and 24 h post-infection, 277 and 900 genes (RSV/control ratio ≥2.0 or ≤0.5), and 1 and 12 pathways respectively were significantly altered. Twenty-three and 92 genes at 4 and 24 h respectively matched respiratory disease biomarkers with ARG2 flagged at 24 h and SCNN1G, EPB41L4B, CSF1, PTEN, TUBB1 and ESR2 at both time points. Hierachical clustering showed a cluster containing ARG2 and IL8. In human bronchial epithelial cells, RSV upregulated arginase II protein. Knockdown of ARG2 increased RSV-induced IL-8, LDH and histone release. With microarray, we identified novel proximal airway epithelial cell genes that may be tested in the sputum samples as biomarkers of RSV infection.     

Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.     

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Δψm collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-κB activation and intracellular Ca²⁺ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.     

CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1.

One neuronal cell line (SK-N-MC) was found to be susceptible to productive infection by multiple isolates of the human immunodeficiency virus type 1 (HIV-1). Characterization of SK-N-MC cells showed that these cells are neuroectodermal in origin in that they express dopamine hydroxylase, catecholamines, neuron-specific enolase, and neurofilaments. Despite their susceptibility to HIV-1 infection, SK-N-MC cells had no detectable CD4 and this infection was not blocked by anti-CD4 monoclonal antibodies (OKT4A, Leu3A) or recombinant soluble CD4. These experiments demonstrated that certain cells of neuroectodermal origin are susceptible to infection in vitro by HIV-1 via a CD4-independent mechanism.     

MicroRNA profile analysis of a feline kidney cell line before and after infection with mink enteritis virus

MicroRNAs (miRNAs) are small regulatory RNAs that play a significant role in eukaryotes by targeting mRNAs for cleavage or translational repression. Recent studies have also shown them to be associated with cellular changes following viral infection. Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. To study the involvement of miRNAs in the MEV infection process, we used Illumina's ultrahigh throughput approach to sequencing miRNA libraries from the feline kidney (F81) cell line before and after infection with MEV. Using this bioinformatics approach we identified 196 known mammalian miRNA orthologs belonging to 152 miRNA families in F81 cells. Additionally, 97 miRNA*s of these miRNAs were detected. As well as known miRNAs, 384 and 398 novel miRNA precursor candidates were identified in uninfected and MEV-infected F81 cells respectively that have not been reported in other mammals. In MEV-infected cells 3 miRNAs were significantly down-regulated and 4 up-regulated including 3 significantly. The majority (12 of 16) of randomly selected miRNA expression profiles by qRT-PCR were consistent with those identified by deep sequencing. A total of 88 miRNAs were predicted to target interferon-associated genes; 6 appear to target the 3′UTR of MEV-specific receptor transferring receptor mRNAs; and 8 to target the MEV mRNA coding region. No miRNAs coded by MEV itself were detected.     

Identification of functional cAMP-dependent protein kinase in a 'neurite minus' mouse neuroblastoma cell line

We have characterized and quantitated the level of cAMP-dependent protein kinase in the NS-20, N1E-115, N-18 and N1A-103 mouse neuroblastoma clonal cell lines, and we have correlated the occurrence of functional cAMP-dependent protein kinase with the dibutyryl cAMP-induced differentiated functions in these cells. Our results demonstrate the presence of functional cAMP-dependent protein kinase in extracts of all four cell lines examined, including the 'neurite minus' N1A-103 cell line. Dibutyryl cAMP induced neurite outgrowth and acetylcholinesterase activity in the NS-20, N1E-115 and N-18 neuroblastoma cell lines, but not in the N1A-103 cell line. However, dibutyryl cAMP caused a 2-3-fold increase in the R1 regulatory subunit protein and cAMP-phosphodiesterase activity in the 'neurite minus' N1A-103 cells in a manner similar to that of the other three 'neurite positive' cell lines. These results suggest that the biochemical lesion(s) subserving the neurite-minus phenotype of the N1A-103 cells may be distal to the activation of cAMP-dependent protein kinase and is in a biochemical pathway distinct from the induction of R1 regulatory subunit protein and cAMP-phosphodiesterase activity.     

DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga.

A DNA methyltransferase was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1. The enzyme recognized the sequence GATC and methylated deoxyadenosine solely in GATC sequences. Host DNA, which contains GATC sequences, but not PBCV-1 DNA, which contains GmATC sequences, was a good substrate for the enzyme in vitro. The DNA methyltransferase activity was first detected about 1 h after viral infection; PBCV-1 DNA synthesis and host DNA degradation also began at about this time. The appearance of the DNA methyltransferase activity required de novo protein synthesis, and the enzyme was probably virus encoded. Methylation of DNAs with the PBCV-1-induced methyltransferase conferred resistance of the DNAs to a PBCV-1-induced restriction endonuclease enzyme described previously (Y. Xia, D. E. Burbank, L. Uher, D. Rabussay, and J. L. Van Etten, Mol. Cell. Biol. 6:1430-1439). We propose that the PBCV-1-induced methyltransferase protects viral DNA from the PBCV-1-induced restriction endonuclease and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.     

Identification and characterization of a DNase induced by Epstein-Barr virus.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.     

Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line

Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 ± 0.40 versus 3.95 ± 0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems. Daniel M. Tiago and Vincent Laizé1 contributed equally to this work.     

Temperature-dependency of Betanodavirus infection in SSN-1 cell line

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.     

Identification of infectious salmon anaemia virus in Atlantic salmon from Nova Scotia (Canada): evidence for functional strain differences

Infectious salmon anaemia (ISA) is a serious disease responsible for high morbidity in farmed Atlantic salmon Salmo salar in Norway, Scotland and New Brunswick, Canada. Recent attempts to identify different strains of ISA virus (ISAV) based on nucleotide sequence variation have shown that the Norwegian and Scottish samples are similar to one another but markedly different from New Brunswick samples. These data may suggest the presence of different strains on each side of the Atlantic but no functional difference has been found with either strain. We describe the first identification and characterisation of ISAV in Atlantic salmon from Nova Scotia, Canada. Further, salmon infected with the Nova Scotia ISAV do not show typical ISAV pathology or mortality. Sequencing of this new strain showed it to possess greater similarity to ISAV from Norway and Scotland than to ISAV from New Brunswick. These findings are discussed in terms of a possible origin of the Nova Scotia ISAV strain and the existence of an avirulent ISAV strain. The impact of current strain variation studies on our knowledge of ISAV is also discussed.     

Intracerebral hemorrhages and syncytium formation induced by endothelial cell infection with a murine leukemia virus.

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.     

Characterization of a porcine kidney cell line resistant to influenza virus infection.

A mutant cell line of porcine kidney cells that resists the cytopathic effect of influenza virus has been obtained and characterized. These cells, designated ESK-R, were originally obtained by prolonged cultivation of cells surviving influenza B/Kanagawa/73 virus infection. No infectious virus was recovered from ESK-R cells, and no evidence for the presence of virus antigens in the cells was demonstrated by immunofluorescent staining. ESK-R cells also showed a distinct resistance to various other strains of both types A and B influenza viruses. The growth of mumps, Sendai, or Newcastle disease virus was considerably restricted, but the cell line normally supported the replication of vesicular stomatitis virus. ESK-R cells were found to lack specific receptors for influenza virus as determined by fluorescence-activated cell sorter analyses. The membrane barrier of ESK-R cells was successfully overcome by nonspecific endocytosis of calcium-coprecipitated virus particles followed by production of an appreciable amount of progeny virus.     

High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus.

Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.     

Theiler's murine encephalomyelitis virus subgroup strain-specific infection in a murine macrophage-like cell line.

We compared infection of a murine macrophage-like cell line, J774-1, with two Theiler's murine encephalomyelitis virus subgroup strains. The GDVII strain, which is highly virulent and produces acute polioencephalomyelitis in mice, did not actively replicate in J774-1 cells, although there was a significant inhibition in cellular protein synthesis. In contrast, the DA strain, which is less virulent and causes demyelination with a persistent virus infection, productively infected J774-1 cells; however, there was less virus produced than in BHK-21 cells, and there was little if any cellular protein shutoff. These in vitro data may provide some explanation for the biological activities that are observed between both subgroup strains.