A cell culture system and production of an infectious clone of Rhopalosiphum padi virus (Dicistroviridae)

Rhopalosiphum padi virus (RhPV) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. RhPV, an icosahedral virus, belongs to the family Dicistroviridae. It has a 10 kb positive-sense RNA genome, with two viral open reading frames (ORFs). We have identified two cell lines, Z10-2 and DMII derived from the homopterans, Homalodisca coagulata and Dalbulus maidis that are permissive for RhPV. Infection, viral replication and production of virions was confirmed by northern blot hybridization, RT-PCR, western blot analysis and immunoelectron microscopy. A cell culture system that allows replication of the virus is crucial for further study of the biology of RhPV;The long term goal of this project is to produce aphid resistant transgenic plants that express RhPV. Acquisition of an infectious clone of RhPV is of primary importance for development of such an aphid resistance technology. A full-length cDNA clone of RhPV was constructed using overlapping reverse transcription PCR products. RNA transcribed from the full-length cDNA clone was infectious upon transfection into Z10-2 and DMII cells, resulting in production of progeny virus phenotypically indistinguishable from the parent virus. The in vitro transcript caused the same cytopathic effects as those caused by transfection of cells with the viral RNA. The virus-like particles (VLPs) purified from the cells transfected with the full-length transcript were infectious to Z10-2 cells and also to R. padi. The infectious cDNA clone of RhPV, together with the cell culture system, will provide valuable experimental tools for the study of replication and pathogenesis of RhPV;The recombinant baculovirus AcRhPV6, which contains the cDNA of RhPV under the control of the polyhedrin promoter, was constructed using the Bac-to-Bac baculovirus expression system. Expression of the AcRhPV6 in Sf21 cells resulted in formation of RhPV VLPs, whose capsids are structurally and immunologically indistinguishable from the native virions. These results show that recombinant baculoviruses can be used to generate immunogenic VLPs of RhPV with some degree of infectivity to aphids. The baculovirus expression system represents an alternative tool for use in the study of the assembly and structure of viruses

Evaluation of Reverse Transcriptase 5\u27 Nuclease Polymerase Chain Reaction assay for the Detection of Viable Heat-Injured and Resuscitated Listeria monocytogenes in Ground Pork

An anaerobic resuscitation-enrichment system was combined with a 5\u27 nuclease reverse transcriptase (RT) protocol for detecting Listeria monocytogenes Scott A from artificially inoculated ground pork. When irradiation-sterilized ground pork containing L. monocytogenes (~6 x 10 5 CFU/g) was heated (60 o C, 14 min), 100% of the cells were injured, as indicated by no growth on selective Modified Oxford (MOX) agar plates incubated aerobically. After resuscitation and enrichment (37°C) in anaerobic Penn State University (PSU) broth, L. monocytogenes was detected within 24 hours both by plating to MOX agar incubated in air and by a fluorogenic 5\u27 nuclease real-time RT-PCR assay. The RT-5\u27 nuclease polymerase chain reaction (PCR) assay targeting the hemolysin gene (hlyA) detected viable L. monocytogenes directly from the PSU within 24 hours, although a stronger signal was detected after 48 hours of resucitation. The RT-5\u27 nuclease PCR assay bypassed the need for subsequent plating of ground pork to selective agar and thus may shorten the interval to detect low numbers of viable L. monocytogenes following heating of naturally contaminated meat

Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

AbstractAvailability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses

The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite.

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors

Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production

Alveolar macrophage-derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes

Type I interferons (IFNs) are important for host defense from viral infections, acting to restrict viral production in infected cells and to promote antiviral immune responses. However, the type I IFN system has also been associated with severe lung inflammatory disease in response to respiratory syncytial virus (RSV). Which cells produce type I IFNs upon RSV infection and how this directs immune responses to the virus, and potentially results in pathological inflammation, is unclear. Here, we show that alveolar macrophages (AMs) are the major source of type I IFNs upon RSV infection in mice. AMs detect RSV via mitochondrial antiviral signaling protein (MAVS)–coupled retinoic acid–inducible gene 1 (RIG-I)–like receptors (RLRs), and loss of MAVS greatly compromises innate immune restriction of RSV. This is largely attributable to loss of type I IFN–dependent induction of monocyte chemoattractants and subsequent reduced recruitment of inflammatory monocytes (infMo) to the lungs. Notably, the latter have potent antiviral activity and are essential to control infection and lessen disease severity. Thus, infMo recruitment constitutes an important and hitherto underappreciated, cell-extrinsic mechanism of type I IFN–mediated antiviral activity. Dysregulation of this system of host antiviral defense may underlie the development of RSV-induced severe lung inflammation

A cell culture system and production of an infectious clone of Rhopalosiphum padi virus (Dicistroviridae)

Rhopalosiphum padi virus (RhPV) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. RhPV, an icosahedral virus, belongs to the family Dicistroviridae. It has a 10 kb positive-sense RNA genome, with two viral open reading frames (ORFs). We have identified two cell lines, Z10-2 and DMII derived from the homopterans, Homalodisca coagulata and Dalbulus maidis that are permissive for RhPV. Infection, viral replication and production of virions was confirmed by northern blot hybridization, RT-PCR, western blot analysis and immunoelectron microscopy. A cell culture system that allows replication of the virus is crucial for further study of the biology of RhPV;The long term goal of this project is to produce aphid resistant transgenic plants that express RhPV. Acquisition of an infectious clone of RhPV is of primary importance for development of such an aphid resistance technology. A full-length cDNA clone of RhPV was constructed using overlapping reverse transcription PCR products. RNA transcribed from the full-length cDNA clone was infectious upon transfection into Z10-2 and DMII cells, resulting in production of progeny virus phenotypically indistinguishable from the parent virus. The in vitro transcript caused the same cytopathic effects as those caused by transfection of cells with the viral RNA. The virus-like particles (VLPs) purified from the cells transfected with the full-length transcript were infectious to Z10-2 cells and also to R. padi. The infectious cDNA clone of RhPV, together with the cell culture system, will provide valuable experimental tools for the study of replication and pathogenesis of RhPV;The recombinant baculovirus AcRhPV6, which contains the cDNA of RhPV under the control of the polyhedrin promoter, was constructed using the Bac-to-Bac baculovirus expression system. Expression of the AcRhPV6 in Sf21 cells resulted in formation of RhPV VLPs, whose capsids are structurally and immunologically indistinguishable from the native virions. These results show that recombinant baculoviruses can be used to generate immunogenic VLPs of RhPV with some degree of infectivity to aphids. The baculovirus expression system represents an alternative tool for use in the study of the assembly and structure of viruses.</p

Evaluation of Reverse Transcriptase 5' Nuclease Polymerase Chain Reaction assay for the Detection of Viable Heat-Injured and Resuscitated Listeria monocytogenes in Ground Pork

An anaerobic resuscitation-enrichment system was combined with a 5' nuclease reverse transcriptase (RT) protocol for detecting Listeria monocytogenes Scott A from artificially inoculated ground pork. When irradiation-sterilized ground pork containing L. monocytogenes (~6 x 10 5 CFU/g) was heated (60 o C, 14 min), 100% of the cells were injured, as indicated by no growth on selective Modified Oxford (MOX) agar plates incubated aerobically. After resuscitation and enrichment (37°C) in anaerobic Penn State University (PSU) broth, L. monocytogenes was detected within 24 hours both by plating to MOX agar incubated in air and by a fluorogenic 5' nuclease real-time RT-PCR assay. The RT-5' nuclease polymerase chain reaction (PCR) assay targeting the hemolysin gene (hlyA) detected viable L. monocytogenes directly from the PSU within 24 hours, although a stronger signal was detected after 48 hours of resucitation. The RT-5' nuclease PCR assay bypassed the need for subsequent plating of ground pork to selective agar and thus may shorten the interval to detect low numbers of viable L. monocytogenes following heating of naturally contaminated meat.</p