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1.
PLoS One ; 11(7): e0160389, 2016.
Article in English | MEDLINE | ID: mdl-27472489

ABSTRACT

The Indianmeal moth, Plodia interpunctella (Lepidoptera: Pyralidae), is a common pest of stored goods with a worldwide distribution. The complete genome sequence for a larval pathogen of this moth, the baculovirus Plodia interpunctella granulovirus (PiGV), was determined by next-generation sequencing. The PiGV genome was found to be 112, 536 bp in length with a 44.2% G+C nucleotide distribution. A total of 123 open reading frames (ORFs) and seven homologous regions (hrs) were identified and annotated. Phylogenetic inference using concatenated alignments of 36 baculovirus core genes placed PiGV in the "b" clade of viruses from genus Betabaculovirus with a branch length suggesting that PiGV represents a distinct betabaculovirus species. In addition to the baculovirus core genes and orthologues of other genes found in other betabaculovirus genomes, the PiGV genome sequence contained orthologues of the bidensovirus NS3 gene, as well as ORFs that occur in alphabaculoviruses but not betabaculoviruses. While PiGV contained an orthologue of inhibitor of apoptosis-5 (iap-5), an orthologue of inhibitor of apoptosis-3 (iap-3) was not present. Instead, the PiGV sequence contained an ORF (PiGV ORF81) encoding an IAP homologue with sequence similarity to insect cellular IAPs, but not to viral IAPs. Phylogenetic analysis of baculovirus and insect IAP amino acid sequences suggested that the baculovirus IAP-3 genes and the PiGV ORF81 IAP homologue represent different lineages arising from more than one acquisition event. The presence of genes from other sources in the PiGV genome highlights the extent to which baculovirus gene content is shaped by horizontal gene transfer.


Subject(s)
Gene Transfer, Horizontal , Genes, Viral , Granulovirus/genetics , Inhibitor of Apoptosis Proteins/genetics , Amino Acid Sequence , Open Reading Frames , Phylogeny , Sequence Homology, Amino Acid
2.
J Gen Virol ; 93(Pt 3): 494-503, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22090214

ABSTRACT

Human coronavirus strain 229E (HCoV-229E) commonly causes upper respiratory tract infections. However, lower respiratory tract infections can occur in some individuals, indicating that cells in the distal lung are susceptible to HCoV-229E. This study determined the virus susceptibility of primary cultures of human alveolar epithelial cells and alveolar macrophages (AMs). Fluorescent antibody staining indicated that HCoV-229E could readily infect AMs, but no evidence was found for infection in differentiated alveolar epithelial type II cells and only a very low level of infection in type II cells transitioning to the type I-like cell phenotype. However, a human bronchial epithelial cell line (16HBE) was readily infected. The innate immune response of AMs to HCoV-229E infection was evaluated for cytokine production and interferon (IFN) gene expression. AMs secreted significant amounts of tumour necrosis factor alpha (TNF-α), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein 1ß (MIP-1ß/CCL4) in response to HCoV-229E infection, but these cells exhibited no detectable increase in IFN-ß or interleukin-29 in mRNA levels. AMs from smokers had reduced secretion of TNF-α compared with non-smokers in response to HCoV-229E infection. Surfactant protein A (SP-A) and SP-D are part of the innate immune system in the distal lung. Both surfactant proteins bound to HCoV-229E, and pre-treatment of HCoV-229E with SP-A or SP-D inhibited infection of 16HBE cells. In contrast, there was a modest reduction in infection in AMs by SP-A, but not by SP-D. In summary, AMs are an important target for HCoV-229E, and they can mount a pro-inflammatory innate immune response to infection.


Subject(s)
Coronavirus 229E, Human/pathogenicity , Macrophages, Alveolar/virology , Cells, Cultured , Cytokines/biosynthesis , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/virology , Fluorescent Antibody Technique, Direct , Gene Expression , Gene Expression Profiling , Humans , Macrophages, Alveolar/immunology , Viral Plaque Assay
3.
J Gen Virol ; 90(Pt 12): 2956-2964, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19741068

ABSTRACT

The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6-8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host.


Subject(s)
Coronavirus Infections , Coronavirus, Rat/pathogenicity , Epithelial Cells/virology , Lung/virology , Pulmonary Alveoli/virology , Virus Replication , Animals , Coronavirus Infections/immunology , Coronavirus Infections/physiopathology , Coronavirus Infections/virology , Coronavirus, Rat/physiology , Cytokines/metabolism , Immunity, Innate , Lung/cytology , Male , Pulmonary Alveoli/cytology , Pulmonary Surfactants/metabolism , Rats , Rats, Inbred F344 , Weight Loss
4.
J Immunol ; 182(3): 1296-304, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19155475

ABSTRACT

Alveolar type II epithelial cells (ATIIs) are one of the primary targets for influenza A pneumonia. The lack of a culture system for maintaining differentiated ATIIs hinders our understanding of pulmonary innate immunity during viral infection. We studied influenza A virus (IAV)-induced innate immune responses in differentiated primary human ATIIs and alveolar macrophages (AMs). Our results indicate that ATIIs, but not AMs, support productive IAV infection. Viral infection elicited strong inflammatory chemokine and cytokine responses in ATIIs, including secretion of IL-8, IL-6, MCP-1, RANTES, and MIP-1beta, but not TNF-alpha, whereas AMs secreted TNF-alpha as well as other cytokines in response to infection. Wild-type virus A/PR/8/34 induced a greater cytokine response than reassortant PR/8 virus, A/Phil/82, despite similar levels of replication. IAV infection increased mRNA expression of IFN genes IFN-beta, IL-29 (IFN-lambda1), and IL-28A (IFN-lambda2). The major IFN protein secreted by type II cells was IL-29 and ATIIs appear to be a major resource for production of IL-29. Administration of IL-29 and IFN-beta before infection significantly reduced the release of infectious viral particles and CXC and CC chemokines. IL-29 treatment of type II cells induced mRNA expression of antiviral genes MX1, OAS, and ISG56 but not IFN-beta. IL-29 induced a dose-dependent decrease of viral nucleoprotein and an increase of antiviral genes but not IFN-beta. These results suggest that IL-29 exerts IFN-beta-independent protection in type II cells through direct activation of antiviral genes during IAV infection.


Subject(s)
Antiviral Agents/metabolism , Cell Differentiation/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Interleukins/metabolism , Macrophages, Alveolar/immunology , Macrophages, Alveolar/virology , Pulmonary Alveoli/immunology , Pulmonary Alveoli/virology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Cell Differentiation/genetics , Cells, Cultured , Chickens , Female , Gene Expression Regulation, Viral/immunology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Interferon-beta/genetics , Interferon-beta/metabolism , Interferons , Interleukins/genetics , Macrophages, Alveolar/cytology , Macrophages, Alveolar/metabolism , Male , Middle Aged , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism
5.
Blood ; 113(10): 2324-35, 2009 Mar 05.
Article in English | MEDLINE | ID: mdl-19104081

ABSTRACT

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.


Subject(s)
Inflammation/immunology , Interleukin-1beta/metabolism , Macrophages/immunology , Monocytes/metabolism , Adenosine Triphosphate/immunology , Adenosine Triphosphate/metabolism , Animals , Blotting, Western , CARD Signaling Adaptor Proteins , CHO Cells , Carrier Proteins/immunology , Carrier Proteins/metabolism , Caspase 1/immunology , Caspase 1/metabolism , Cricetinae , Cricetulus , Cytoskeletal Proteins/immunology , Cytoskeletal Proteins/metabolism , Enzyme Activation/immunology , Humans , Lipopolysaccharides/immunology , Monocytes/immunology , NLR Family, Pyrin Domain-Containing 3 Protein , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction
6.
Virology ; 372(1): 127-35, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-18022664

ABSTRACT

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10(5) pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.


Subject(s)
Epithelium/virology , Pulmonary Alveoli/cytology , Pulmonary Alveoli/virology , Severe acute respiratory syndrome-related coronavirus/physiology , Virus Replication , Cell Differentiation , Cells, Cultured , Cytopathogenic Effect, Viral , Humans , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/genetics , Virology/methods
7.
Am J Respir Cell Mol Biol ; 36(6): 661-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17255555

ABSTRACT

Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.


Subject(s)
Cell Culture Techniques , Cell Differentiation/physiology , Epithelial Cells , Pulmonary Alveoli/cytology , Animals , Biomarkers/metabolism , Cell Polarity , Cell Shape , Cells, Cultured , Collagen/metabolism , Drug Combinations , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Laminin/metabolism , Lipids/biosynthesis , Phenotype , Proteoglycans/metabolism , Rats
8.
Anal Biochem ; 329(1): 68-76, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15136168

ABSTRACT

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4-14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 microM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.


Subject(s)
Adenosine Triphosphate/metabolism , Biological Assay/methods , Kinesins/metabolism , Humans , Kinesins/antagonists & inhibitors , Kinetics , Molecular Motor Proteins/agonists , Molecular Motor Proteins/antagonists & inhibitors , Molecular Motor Proteins/metabolism , Pyrimidines , Solvents , Thiones
9.
Curr Microbiol ; 48(1): 77-81, 2004 Jan.
Article in English | MEDLINE | ID: mdl-15018108

ABSTRACT

The whitefly Bemisia tabaci contains a primary prokaryotic endosymbiont housed within specialized cells in the body cavity. Two DNA fragments from the endosymbiont, totaling 33.3 kilobases, were cloned and sequenced. In total, 37 genes were detected and included the ribosomal RNA operon and genes for ribosomal RNA proteins. The guanine plus cytosine of the DNA was 30.2 mol%, different from that of endosymbionts of other plant sap-sucking insects.


Subject(s)
Hemiptera/microbiology , Proteobacteria/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Symbiosis/genetics
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