Assessing protection against OP pesticides and nerve agents provided by wild-type HuPON1 purified from Trichoplusia ni larvae or induced via adenoviral infection

2013 ◽  
Vol 203 (1) ◽  
pp. 177-180 ◽  
Author(s):  
Sean M. Hodgins ◽  
Shane A. Kasten ◽  
Joshua Harrison ◽  
Tamara C. Otto ◽  
Zeke P. Oliver ◽  
...  
Keyword(s):  
Trichoplusia Ni ◽  
Nerve Agents ◽  
Wild Type ◽  
Adenoviral Infection

scholarly journals Effects of Ac150 on virulence and pathogenesis of Autographa californica multiple nucleopolyhedrovirus in noctuid hosts

2005 ◽  
Vol 86 (6) ◽  
pp. 1619-1627 ◽  
Author(s):  
Ji-Hong Zhang ◽  
Taro Ohkawa ◽  
Jan O. Washburn ◽  
Loy E. Volkman
Keyword(s):  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Lepidopteran Insect ◽  
Charged Amino Acids ◽  
Multiple Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

Ac150 is expressed late during infection of cultured lepidopteran insect cells by Autographa californica multiple nucleopolyhedrovirus. The Ac150 gene product is predicted to have a molecular mass of 11 161 Da and consists of a hydrophobic N terminus and a single ‘peritrophin-A’-like domain, connected by a short region of charged amino acids. An Ac150 deletion mutant and its parental wild-type virus were compared for differences in virulence by both oral and intrahaemocoelic routes of infection. It was found that the mutant was significantly less virulent in larvae of all three host species tested (Heliothis virescens, Spodoptera exigua and Trichoplusia ni) when occlusions were administered orally, but not when isolated occlusion-derived virus (ODV) was administered orally or budded virus was administered intrahaemocoelically. ODV yields were the same from equal numbers of mutant and wild-type occlusions, and nucleocapsid-distribution frequencies within the two ODV populations were the same, eliminating these features as explanations for the observed differences in virulence. Comparison of pathogenesis, as revealed by lacZ expression from identical reporter-gene cassettes in the mutant and wild-type virus, indicated that the mutant was less efficient at establishing primary infection in midgut cells; otherwise, it exhibited infection kinetics identical to those of wild-type virus. Ac150, therefore, can be considered a per os infection factor that mediates, but is not essential for, oral infection.

scholarly journals A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

2003 ◽  
Vol 69 (4) ◽  
pp. 2052-2057 ◽  
Author(s):  
James C. Bull ◽  
H. C. J. Godfray ◽  
David R. O'Reilly
Keyword(s):  
Cell Culture ◽  
Population Genetics ◽  
Trichoplusia Ni ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Cell Infection ◽  
Occlusion Bodies ◽  
Budded Virus

ABSTRACT Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

scholarly journals Both Lymantria disparNucleopolyhedrovirus Enhancin Genes Contribute to Viral Potency

2001 ◽  
Vol 75 (18) ◽  
pp. 8639-8648 ◽  
Author(s):  
Holly J. R. Popham ◽  
David S. Bischoff ◽  
James M. Slavicek
Keyword(s):  
Trichoplusia Ni ◽  
Amino Acid Identity ◽  
Wild Type Virus ◽  
Northern Analysis ◽  
Gene Transcript ◽  
Type Virus ◽  
Wild Type ◽  
Entire Genome ◽  
Average Decrease ◽  
Gene Transcripts

ABSTRACT Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified anenhancin gene (E1) in the Lymantria disparmultinucleocapsid nucleopolyhedrovirus (LdMNPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133–8140, 1997). Inactivation of the E1 gene product within the viral genome lowered viral potency by an average of 2.9-fold. A secondenhancin gene (E2) was identified when the entire genome of LdMNPV was sequenced (Kuzio et al., Virology 253:17–34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the LdMNPV E1 protein as well as the enhancins fromTrichoplusia ni GV, Pseudaletia unipuncta GV,Helicoverpa armigera GV, and Xestia c-nigrumGV. Northern analysis of viral RNA indicated that the E2gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the E2 gene (E2del) and a second with deletion mutations in bothenhancin genes (E1delE2del). Theenhancin gene viral constructs were verified by Southern analysis and shown not to produce enhancin gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce anE1 gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both LdMNPV enhancin genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency.

scholarly journals Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

2001 ◽  
Vol 67 (3) ◽  
pp. 1140-1146 ◽  
Author(s):  
Pedro Hernández-Crespo ◽  
Steven M. Sait ◽  
Rosemary S. Hails ◽  
Jenny S. Cory
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Genetically Modified ◽  
Recombinant Baculovirus ◽  
Virus Yield ◽  
Wild Type Virus ◽  
Mamestra Brassicae ◽  
Speed Of Kill ◽  
Wild Type ◽  
Susceptible Host

ABSTRACT Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae ofM. brassicae. Both viruses produced a greater yield inM. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.

scholarly journals Caspase inhibitor P35 is required for the production of robust baculovirus virions in Trichoplusia ni TN-368 cells

2009 ◽  
Vol 90 (3) ◽  
pp. 654-661 ◽  
Author(s):  
Bart Bryant ◽  
Rollie J. Clem
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Mutant Virus ◽  
Progeny Virus ◽  
Caspase Inhibitor ◽  
Wild Type ◽  
Lepidopteran Insects ◽  
Baculovirus Infection

Apoptosis can protect lepidopteran insects against baculovirus infection by limiting viral replication. Baculoviruses counter this response by expressing anti-apoptotic proteins such as the caspase inhibitor P35, which is expressed by several baculoviruses including Autographa californica mutiple nucleopolyhedrovirus (AcMNPV). Mutants of AcMNPV that lack the p35 gene induce apoptosis in Spodoptera frugiperda cells, and replication of these mutants is severely curtailed in S. frugiperda cell lines and larvae. However, cells from another lepidopteran species, Trichoplusia ni, do not undergo apoptosis when infected with AcMNPV mutants lacking p35, and p35 mutant and wild-type viruses replicate to equivalent levels in the T. ni cell line TN-368 and have equivalent infectivity in T. ni larvae by either oral or intrahaemocoelic injection. This has led to the conclusion that p35 is not required for AcMNPV replication in T. ni. However, in this study it was found that stocks of p35 mutant virus produced in TN-368 cells had defects in virion stability and infectivity. TN-368 cells infected with p35 mutant AcMNPV exhibited caspase activity, despite a lack of apoptosis, and propagation of the mutant virus in the presence of a chemical caspase inhibitor restored the normal infection phenotype to the progeny virus. These results suggest that caspases can directly or indirectly damage baculovirus virions, and reveal a novel aspect of the role of apoptosis in antiviral defence.

scholarly journals Baculovirus lef-12 Is Not Required for Viral Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12032-12043 ◽  
Author(s):  
Linda A. Guarino ◽  
Toni-Ann Mistretta ◽  
Wen Dong
Keyword(s):  
Trichoplusia Ni ◽  
Late Phase ◽  
Growth Curves ◽  
Single Step ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Step Growth

ABSTRACT The baculovirus lef-12 (orf41) gene is required for transient expression of baculovirus late genes. To analyze the role of LEF-12 in the context of infected cells, two mutant viruses were constructed. Both mutants were viable in Trichoplusia ni High 5 and Spodoptera frugiperda Sf9 cells. Single-step growth curves, however, indicated that virus yields were reduced approximately fivefold in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to or only twofold lower than those of wild-type virus-infected cells. Western blot analyses confirmed that LEF-12 was not synthesized in cells infected with mutant virus. In wild-type virus-infected cells, LEF-12 was not detected until 18 h postinfection, and accumulation of LEF-12 peaked at 24 to 36 h postinfection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 h postinfection and peaked between 18 and 24 h postinfection. Furthermore, synthesis of lef-12 mRNA and LEF-12 protein were inhibited by the addition of aphidicolin, indicating that lef-12 is expressed after DNA replication.

Clara cell secretory protein decreases lung inflammation after acute virus infection

1998 ◽  
Vol 275 (5) ◽  
pp. L924-L930 ◽  
Author(s):  
Kevin S. Harrod ◽  
Amber D. Mounday ◽  
Barry R. Stripp ◽  
Jeffrey A. Whitsett
Keyword(s):  
Viral Infection ◽  
Lung Inflammation ◽  
Cell Injury ◽  
Pulmonary Inflammation ◽  
Secretory Protein ◽  
Clara Cell ◽  
Wild Type ◽  
Monocyte Chemoattractant Protein 1 ◽  
Adenoviral Infection ◽  
Clara Cell Secretory Protein

Clara cell secretory protein (CCSP) is an abundant 10-kDa polypeptide synthesized and secreted primarily by nonciliated bronchiolar epithelial cells in the mammalian lung. To determine the potential role of CCSP in pulmonary inflammation after acute viral infection, CCSP gene-targeted {CCSP-deficient [CCSP(−/−)]} mice were exposed to a recombinant E1- and E3-deficient adenoviral vector, Av1Luc1, intratracheally. Lung inflammation was markedly increased in CCSP(−/−) mice compared with wild-type control mice and was associated with an increased number of polymorphonuclear cell infiltrates and epithelial cell injury in both conducting airways and alveolar regions. Histological evidence of pulmonary inflammation in CCSP(−/−) mice was associated with increased production of cytokine (interleukin-1β and -6 and tumor necrosis factor-α) mRNA and protein, as well as chemokine (macrophage inflammatory protein-1α and -2 and monocyte chemoattractant protein-1) mRNA expression within the lung in response to adenoviral infection. Adenoviral-mediated gene transfer was decreased in CCSP(−/−) mice relative to wild-type mice as measured by luciferase enzyme activity in lung homogenates. The present study suggests that CCSP is involved in modulating lung inflammation during viral infection and supports a role for CCSP in lung host defense.

scholarly journals Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

2021 ◽  
Author(s):  
Pei-Ju Liu ◽  
Laura K Gunther ◽  
Diana Perez ◽  
Jing Bi-Karchin ◽  
Christopher D Pellenz ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Protein Sequence ◽  
Expression System ◽  
Cell Junctions ◽  
Wild Type ◽  
Steroid Resistant ◽  
Steroid Resistant Nephrotic Syndrome ◽  
Adenoviral Infection

Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

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