Nodamura virus nonstructural protein B2 can enhance viral RNA accumulation in both mammalian and insect cells

During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.     

Transition metal complexes IX. Nickel complexes with a chiral azaphosphole ligand

The reaction of (−)-(R)-myrtenal and (+)-(R)-phenylethylamine gave a Schiff base 1 which was reacted with MePBr₂ in the presence of a base to give under dehydrohalogenation of an intermediate McCormack product a salt 2. Treatment of 2 with sodium led to the formation of the azaphosphole 4. η³-C₃H₅NiCl and 4 gave a 1:1 adduct 5 and nickel(0) gave a 1:4 complex 6. Compounds 4–6 were characterized by NMR spectroscopy as well as by single crystal X-ray structure determination.     

Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba''s short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.     

High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants

Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3'-to-5' exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.     

Addition of arthropod cocoons to house wren nests is correlated with delayed pairing

Males in the cavity-nesting house wren (Troglodytes aedon) frequentlyadd arthropod cocoons to their nests during building, possiblyas an ornamental cue for female choice. We tested this hypothesisby comparing the time to pairing for males that did and didnot add cocoons to their nests and for males in whose nestswe manipulated the number of cocoons prior to pairing. We alsotested the hypothesis that females acquire fitness-related benefitsby selecting mates based on their use of cocoons. The use ofcocoons by males was not consistently related to habitat, butthe number of cocoons added per nest increased during the courseof the breeding season. Contrary to prediction, the time topairing for males adding cocoons was significantly longer thanthat for males without cocoons in their nests at both unmanipulatedand experimental nests. There was also no consistent fitness-relatedbenefit for females related to the use of cocoons by their mates.Therefore, we conclude that females did not prefer males thatadded cocoons to their nests, and that the increased time topairing for males that add cocoons likely results in fitness-relatedcosts brought about by delayed breeding. Nonetheless, male housewrens routinely use cocoons, and why they do so remains unknown.     

Nodamura virus RNA replication in Saccharomyces cerevisiae: heterologous gene expression allows replication-dependent colony formation

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.     

Estimating the probability of bacterial infection using a novel biomarker among pediatric patients in the emergency department

Context: IL-27 is a novel biomarker to identify bacterial infection in children.

Objective: IL-27 was evaluated among pediatric emergency department (ED) patients and compared with procalcitonin (PCT).

Methods and results: Children undergoing blood, urine, or cerebrospinal fluid cultures had IL-27 and PCT assays performed. Bacterial infection was defined as a positive culture or a clinical diagnosis based on chart review. IL-27 and PCT were increased among patients with bacterial infection and demonstrated comparable AUC’s (0.62 versus 0.61). A decision tree incorporating IL-27, PCT, and white blood cell count improved the AUC (0.80).

Conclusion: IL-27 is a viable candidate biomarker to identify bacterial infection among ED patients and is comparable with PCT.     

Interferon-resistant Daudi Cell Line with a Stat2 Defect Is Resistant to Apoptosis Induced by Chemotherapeutic Agents

Interferon-α (IFNα) has shown promise in the treatment of various cancers. However, the development of IFN resistance is a significant drawback. Using conditions that mimic in vivo selection of IFN-resistant cells, the RST2 IFN-resistant cell line was isolated from the highly IFN-sensitive Daudi human Burkitt lymphoma cell line. The RST2 cell line was resistant to the antiviral, antiproliferative, and gene-induction actions of IFNα. Although STAT2 mRNA was present, STAT2 protein expression was deficient in RST2 cells. A variant STAT2 mRNA, which resulted from alternative splicing within the intron between exon 19 and 20, was expressed in several human cell lines but at relatively high levels in RST2 cells. Most importantly, the RST2 line showed an intrinsic resistance to apoptosis induced by a number of chemotherapeutic agents (camptothecin, staurosporine, and doxorubicin). Expression of STAT2 in RST2 cells not only rescued their sensitivity to the biological activities of IFNs but also restored sensitivity to apoptosis induced by these chemotherapeutic agents. The intrinsic resistance of the RST2 cells to IFN as well as chemotherapeutic agents adds a new dimension to our knowledge of the role of STAT2 as it relates to not only biological actions of IFN but also resistance to chemotherapy-induced apoptosis.IFN²α/β regulates a number of cellular responses, such as proliferation, differentiation, and development (1). Although IFN triggers the death of some tumor cells by inducing proapoptotic proteins (tumor necrosis factor-related apoptosis-inducing ligand, PKR, etc.), IFN also promotes cell survival through a nuclear factor κB-dependent pathway (2–4). IFN is used to treat various human malignancies (chronic myeloid leukemia, non-Hodgkin lymphomas, Kaposi sarcoma, hairy cell leukemia, multiple myeloma, and malignant melanoma), viral infections, as well as various other diseases (5, 6). However, only a fraction of patients are responsive to IFN therapy, and many patients eventually develop resistance after chronic IFN exposure. The underlying mechanism for IFN resistance is still unclear, but it is reasonable to suggest that genetic variation and selection during prolonged IFN exposure may reflect IFN signaling defects.IFN binds to its cell surface receptor resulting in the activation of JAK1 and TYK2 nonreceptor protein-tyrosine kinases, which phosphorylate STAT proteins (7). Phosphorylated STAT1 and STAT2 in a complex with IRF9 bind to a conserved IFN stimulus-response element (ISRE) present in the promoters of hundreds of IFN-stimulated genes (ISGs) inducing their expression. Mutant cell lines with defined signaling defects have made significant contributions in elucidating the IFN-activated JAK/STAT signal transduction pathway (7, 8). Such IFN-resistant mutants were isolated after multiple rounds of chemical mutagenesis and selection of IFN-resistant mutants. However, this procedure does not mimic in vivo what happens to patients who are subjected to long term IFN treatment. Moreover, these IFN-resistant cells have undergone multiple mutational events, because complementation of a single defect rescues aspects of IFN signaling but not sensitivity to all of the biological actions of IFN (8, 9). As an alternative approach, our laboratory has resorted to long term IFN treatment of cells to isolate naturally arising IFN-resistant mutants, which more closely resemble what occurs in vivo (10, 11). Using this strategy, we previously identified a STAT3-defective IFN-resistant cell line (11).In this study, a mutant cell line (RST2) that was highly resistant to the antiviral, antiproliferative, and gene-inducing actions of IFN was isolated from the highly IFN-sensitive Daudi cell line by growth in the continuous presence of IFN. Sequencing of STAT2 mRNA identified an alternative splice site between exon 19 and 20 that is expressed in RST2 cells, causing translation termination at the beginning of the Src homology 2 domain of STAT2. Expression of STAT2 in RST2 cells rescued sensitivity to the antiviral, antiproliferative, and gene-inducing actions of IFN. Furthermore, although RST2 cells are intrinsically resistant to the induction of apoptosis by a variety of chemotherapeutic agents, reconstitution of STAT2 restored sensitivity to chemotherapy-induced apoptosis.