Silkworm salivary glands are not susceptible to Bombyx mori nuclear polyhedrosis virus

A nuclear polyhedrosis virus isolated from infected Bombyx mori, BmNPV, was used to inoculate silkworms to determine salivary gland cell susceptibility. The salivary gland was removed from infected silkworms at different times post-inoculation and examined by light microscopy. The salivary gland cells did not exhibit any signs of BmNPV infection; however, fat body and tracheal cells, used as positive controls, showed characteristic cytopathological changes caused by BmNPV infection, which confirmed inoculum viability. The morphological distribution of tracheal branches and the basal lamina, which serves as a barrier to viral penetration, are apparently involved in this resistance to infection.     

Eclipse period of baculovirus infection in larvae of the armyworm, Pseudaletia unipuncta

Two strains of a nuclear polyhedrosis virus (baculovirus) infect larvae of the armyworm, Pseudaletia unipuncta. The hypertrophy strain (HNPV) produces a gradient of infected epithelial cells along the tracheae indicating the movement of infectious material to adjacent cells. Cytopathology of the eclipse period up to the appearance of the virogenic stroma has been separated into three phases during which the chromatin disappears and is replaced by dense interconnected strands of fibrils and dense punctate bodies. Cellular hypertrophy occurs in phase 1 and the virogenic stroma appears in phase 3. The typical strain (TNPV) does not produce structures comparable to those of HNPV infection.     

Identification and characterization of a baculovirus occlusion body glycoprotein which resembles spheroidin, an entomopoxvirus protein.

A 37-kDa polypeptide specified by Autographa californica nuclear polyhedrosis virus was found to share significant homology with Choristoneura biennis entomopoxyvirus spheroidin protein, which is the major component of entomopoxvirus occlusion bodies. Antibodies raised against spheroidin cross-reacted with the 37-kDa protein and confirmed its expression in the late phase of wild-type baculovirus infection. Immunoblot analysis and fluorescence microscopy demonstrated that the protein was associated with purified A. californica nuclear polyhedrosis virus occlusion bodies and was absent in purified virions. Immunofluorescence studies localized the protein to the periphery of occlusion bodies and the internal membranes of cells infected with wild-type baculovirus. The open reading frame encoding this spheroidinlike protein was inserted into a baculovirus expression vector, and recombinant protein was synthesized under control of the polyhedrin promoter. Studies of the recombinant protein demonstrated that it was heterogeneous in molecular mass as a result of N-linked glycosylation. Tunicamycin inhibited carbohydrate addition and yielded proteins of 34 and 33 kDa.     

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

Bordetella avium causes induction of apoptosis and nitric oxide synthase in turkey tracheal explant cultures

Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.     

Difference of proteins from inclusion bodies formed in the nucleus and cytoplasm of the cytoplasmic polyhedrosis virus-infected midgut in the silkworm,Bombyx mori

Strains of cytoplasmic polyhedrosis virus (CPV) of the silkworm Bombyx mori typically form proteinaceous inclusion bodies (IBs) which occlude many virions and are formed in the cytoplasm of the midgut epithelium. In contrast, an unusual strain of CPV termed “A” strain produces IBs containing no virions in the nuclei of the epithelial cells. In this case although the viruses multiply in the cytoplasm, few IBs are formed in the cytoplasm. To clarify why the A strain forms IBs in the nucleus, the structural differences on the IB proteins (IBPs) from A and a typical (H) strain were investigated. Analyses by SDS-PAGE showed A strain IBP had slightly lower electrophoretic mobility than these of H strain. When these IBPs were partially digested with Staphylococcus aureus V8 protease, one of the digested products between the two strains showed different electrophoretic mobility. Amino acid sequence analyses of peptides produced with lysylendopeptidase from IBP of A strain indicated that a histidine residue of H strain was replaced by a tyrosine residue. The carboxyl terminal regions of the two IBPs were also different; IBP of A strain was -Leu-Leu-Val-COOH, but the terminal residue of H strain could not be determined by the same method. These differences of amino acid sequence of IBPs between A and H strains may be responsible for the partitioning of them; i.e., in the case of A strain IBP may be transportable into the nucleus by the specific signal of amino acid sequence.     

Ultrastructure of fat body cells of the velvetbean caterpillar infected with a nuclear polyhedrosis virus

During a study of the ultrastructure of a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis, various types of nuclear and cytoplasmic inclusions were found in fat body tissue heavily infected with the virus. Virogenic stroma was present in the nuclei of most infected cells. Bundles of fibrous material were observed in the nuclei and cytoplasm of cells containing polyhedral bodies. Other nuclear inclusions included concentric multilayered material, vacuoles, and membrane structures.     

Enhancing the efficacy and persistency of Spodoptera litura (Fab.) nuclear polyhedrosis virus using UV irradiation protectants

To enhance the field persistency of S. litura nuclear polyhedrosis virus (SLNPV), three chemicals viz. cupric ammonium nitrate, tinopal and cupric sulphate were tried as protectants (0.01 mg/ml) against natural sunlight (UV) irradiation. On exposure for 8 hr and subsequent bioassaying (diet surface treatment), it was found that cupric sulphate protected the polyhedrosis inclusion bodies (PIBs), recording 95.56% mortality which was statistically at par with unexposed PIBs recording 97.78% mortality.     

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.     

Inspiratory rhythm in airway smooth muscle tone

In anesthetized paralyzed open-chested cats ventilated with low tidal volumes at high frequency, we recorded phrenic nerve activity, transpulmonary pressure (TPP), and either the tension in an upper tracheal segment or the impulse activity in a pulmonary branch of the vagus nerve. The TPP and upper tracheal segment tension fluctuated with respiration, with peak pressure and tension paralleling phrenic nerve activity. Increased end-tidal CO2 or stimulation of the carotid chemoreceptors with sodium cyanide increased both TPP and tracheal segment tension during the increased activity of the phrenic nerve. Lowering end-tidal CO2 or hyperinflating the lungs to achieve neural apnea (lack of phrenic activity) caused a decrease in TPP and tracheal segment tension and abolished the inspiratory fluctuations. During neural apnea produced by lowering end-tidal CO2, lung inflation caused no further decrease in tracheal segment tension and TPP. Likewise, stimulation of the cervical sympathetics, which caused a reduction in TPP and tracheal segment tension during normal breathing, caused no further reduction in these parameters when the stimulation occurred during neural apnea. During neural apnea the tracheal segment tension and TPP were the same as those following the transection of the vagi or the administration of atropine (0.5 mg/kg). Numerous fibers in the pulmonary branch of the vagus nerve fired in synchrony with the phrenic nerve. Only these fibers had activity which paralleled changes in TPP and tracheal tension. We propose that the major excitatory input to airway smooth muscle arises from cholinergic nerves that fire during inspiration, which have preganglionic cell bodies in the ventral respiratory group in the region of the nucleus ambiguus and are driven by the same pattern generators that drive the phrenic and inspiratory intercostal motoneurons.     

Central nervous system features of a nicotine-resistant insect, the tobacco hornworm Manduca sexta

The purpose of this study is to look for structural correlates of the demonstrated nicotine-insensitivity of larval Manduca sexta CNS, an insensitivity which is only slightly perturbed by desheathing (a technique used to disrupt perineurial diffusion barriers). The general organization of the hornworm ganglion is found to conform to the conventional insect pattern, but the following points are noted and discussed in terms of their potential relationship to nicotine-insensitivity: the damage caused to perineurial cells by desheathing is extremely localized, with cells immediately adjacent to the torn region showing good ultrastructural integrity; ionic lanthanum does not gain access to the subperineurial extracellular space following desheathing; lanthanum penetrates the ganglion in the cytoplasm of tracheal cells damaged peripherally during desheathing, but is excluded from the extracellular space surrounding such tracheal cells; smooth endoplasmic reticulum is much in evidence in perineurial cells and tracheal cells, sites where it might be implicated in nicotine detoxification; individual basal perineurial cells appear to cover extensive regions of the ganglion, thereby limiting intercellular diffusion.     

Electron microscope observations of Autographa californica (Noctuidae) nuclear polyhedrosis virus replication in the midgut of the saltmarsh caterpillar, Estigmene acrea (Arctiidae)

The nuclear polyhedrosis virus from Autographa californica was studied with the electron microscope in the midgut of the salt marsh caterpillar, Estigmene acrea. The results of the present study were compared with a previous study in which the same inoculum was fed to Spodoptera exigua. In Estigmene acrea polyhedra were produced, but virions were not occluded. Nonoccluded virions were found throughout the midgut cytoplasm and budding into the hemocoel. Within the cytoplasm, the rough endoplasmic reticulum was observed to contain paracrystalline proteinaceous bodies. Fibrous bodies and annulate lamellae were also found in the cytoplasm of infected cells.     

Ionic conditions affecting the release and absorption of an alkaline protease associated with the occlusion bodies of insect baculoviruses

Nearly all of the alkaline protease found in the occlusion bodies of baculoviruses (polyhedra for nuclear polyhedrosis and capsules for granulosis viruses) (Baculovirus, subgroup A and B, family Baculoviridae) can be specifically extracted under high ionic concentration. The extraction is directly proportional to the concentrations of NaCl up to 0.25 m. It is not dependent on pH, species of ions, temperature, and incubation time. The protease is reabsorbed under low ionic concentration by protease-extracted and by heat-treated capsules and polyhedra. The protease from Streptomyces griseus is not absorbed. This indicates that the occlusion body proteins have distinct affinity for certain alkaline proteases.     

Pathology of aspiration pneumonia in mice

A aspiration pneumonia caused in 3 female mice by food inhalation was examined histopathologically. Dark-red areas were observed macroscopically on the uncut lungs. The change was more severe in the superior and post-caval lobes of the right lung and the anterior part of the left lung. Microscopic examination revealed various-sized foreign bodies (inhaled food particles) accompanied by inflammatory cells and microorganisms in the tracheal lumen and the bronchiolar and alveolar spaces.     

Immuno-electron-microscopic localization of a centriole-related antigen in ciliated cells

Summary An antigen common to purported centriolar and basal body regions of a variety of cell types was previously visualized by immuno-fluorescence microscopy. The present study demonstrates the localization of the antigen relative to the defined basal body structures of ciliated tracheal cells at the electron-microscopic level. After ethyldimethylaminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation and permeabilization, immunoferritin labeling is consistently found associated with amorphous electron-opaque material in proximity to basal bodies and their ciliary rootlets, but not with basal body microtubules themselves. This distribution pattern is distinct from that of other proteins found in the apical region of ciliated cells, such as calmodulin. It is proposed that the dense material may be analogous to pericentriolar material of centrosomes.     

Immobilization of bioactive fibroblast growth factor-2 into cubic proteinous microcrystals (Bombyx mori cypovirus polyhedra) that are insoluble in a physiological cellular environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.     

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.     

The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta

The hypertrophy nuclear polyhedrosis virus of the armyworm, Pseudaletia unipuncta, causes a unique gradient of infected cells to form on the trachea. The movement and invasion of the virus apparently were not through adjacent intercellular membranes. The enveloped viruses emerged from the initially infected cell into an area between the cell plasma membrane and basal lamina, and then entered the uninfected tracheal cell either by lateral attachment and fusion of the viral envelope and the plasma membrane or by viropexis. The two methods of viral invasion into the cell suggest the presence of at least two phenotypically different enveloped viruses. Viropexis was initiated with an alignment of the peplomer spikes with regularly spaced, short radial striations on the inner coat of the plasma membrane. At a late state in viropexis, the viral envelope fused with the vacuole membrane, and an opening developed below the site of membrane fusion through which the nucleocapsid might enter the cytoplasm. Some nucleocapsids in membrane-lined vesicles resulting from viropexis appeared to be in a state of dissolution. Naked nucleocapsids were found along the nuclear envelope and within the nucleoplasm. No uncoating of the nucleocapsids was observed at the nucleopores, but uncoating seemed to occur in the nucleoplasm. Nucleocapsids were also found in the cytoplasm of nonsusceptible fat body cells, in which virus replication was not observed.