Infected Midgut Cells Research Articles

Pathogenicity of Serratia marcescens to hazelnut weevil (Curculio dieckmanni)

The hazelnut weevil (Curculio dieckmanni Faust.) is a major pest of Asian hazel (Corylus heterophylla Fisch.) in China. Dead hazelnut weevil larvae were examined and the associated pathogenic bacterium was identified as Serratia marcescens Bizio. This significantly shortened the lifespan of hazelnut weevil. Larval weight was reduced as a function of S. marcescens concentration and exposure time. The structure of infected midgut cells was altered, with necrosis of the wall tissues and many cells becoming dislodged, creating cavities. The S. marcencens strain inhibited digestive enzyme activity and protective enzymes in the midgut of adult hazelnut weevil. Inhibition on S. marcencens strain increased with treatment time. S. marcescens directly destroyed the midgut cells and interfered with digestive and protective enzymes. This decreased the food intake and increased mortality of hazelnut weevil. S. marcescens appears to be an effective bacterium for the control of hazelnut weevil but requires further study, including biological formulation development and field application.

Mamestra configurata nucleopolyhedrovirus-A transcriptome from infected host midgut

Infection of an insect by a baculovirus occurs in two distinct phases, an initial infection of host midgut by occlusion-derived virions (ODVs) and subsequent systemic infection of other tissues by budded virions (BV). A vast majority of investigations of the infection process have been restricted to cell culture studies using BV that emulate the systemic phase of infection. This is one of the first studies to investigate baculovirus gene expression in ODV infected midgut cells. We have focused on the critical first phase of in vivo infection by Mamestra configurata nucleopolyhedrovirus-A in M. configurata larvae, using qPCR and RNAseq mass sequencing to measure virus gene expression in midgut cells. The earliest genes detected by each method had significant overlap, including known early genes as well as genes unique to MacoNPV-A and genes of unknown function. The RNAseq data also revealed a large range of expression levels across all ORFs, which could not be measured using qPCR. This dataset provides a first whole genome transcriptomic analysis of viral genes required for virus infection in vivo and will provide the basis for functionally analyzing specific genes that may be critical elements in baculovirus midgut infectivity and host range.

First Isolation, In Egypt, of a granulovirus (AgseGVEG) from Agrotis segetum Schiffermuller (Lepidoptera: Noctuidae)

A new Granulovirus was isolated from cutworm Agrotis segetum larvae collected from the field in Qalubaya governorate. Transverse section of larvae showed infected fat body, epidermis and mid-gut cells. Transmition electromicroscope evealed ovoid occlusion bodies (granules), each granule contains a single rod-shaped virion with one nucleocapsid, typical of granuolosis virus. Also, the scanning electromicroscopy of apurified viral suspension confirmed a baculovirus morphology. The size of virus particles in the present isolation ranged between 1.9 m to 2.5m. Preliminary bioassay indicated that A. ipsilon neonate larvae are susceptible to AgseGV EG and the calculated LC50 value was 3.79x10 6 capsules/ml.

Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylittacytoplasmic polyhedrosis virus

BackgroundAntheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.ResultsIn this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV), Lymantria dispar CPV (LdCPV), and Dendrolimus punctatus CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.ConclusionOur results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.

Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: Infection initiated by a small number of susceptible epithelial cells and a population bottleneck

We evaluated infection of Aedes taeniorhynchus mosquitoes, vectors of Venezuelan equine encephalitis virus (VEEV), using radiolabeled virus and replicon particles expressing green (GFP) or cherry fluorescent protein (CFP). More epidemic VEEV bound to and infected mosquito midguts compared to an enzootic strain, and a small number of midgut cells was preferentially infected. Chimeric replicons infected midgut cells at rates comparable to those of the structural gene donor. The numbers of midgut cells infected averaged 28, and many infections were initiated in only 1–5 cells. Infection by a mixture of GFP- and CFP-expressing replicons indicated that only about 100 midgut cells were susceptible. Intrathoracic injections yielded similar patterns of replication with both VEEV strains, suggesting that midgut infection is the primary limitation to transmission. These results indicate that the structural proteins determine initial infection of a small number of midgut cells, and that VEEV undergoes population bottlenecks during vector infection.

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Fluorescent brighteners significantly lower the LC50 and LT50 in a variety of nucleopolyhedrovirus–insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24–48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.

Autographa californica M nucleopolyhedrovirus early GP64 synthesis mitigates developmental resistance in orally infected noctuid hosts.

The unusual early synthesis of the Autographa californica M nucleopolyhedrovirus (AcMNPV) budded virus (BV) structural protein GP64 is an important virulence factor during oral infection of Heliothis virescens larvae. Considering the breadth of the AcMNPV host range, the importance of early GP64 synthesis in orally infected permissive hosts (Trichoplusia ni and Spodoptera exigua) from subfamilies other than that of H. virescens was assessed. An AcMNPV recombinant, having wild-type early and late GP64 synthesis, was compared with one in which only late GP64 synthesis occurred. Early GP64 synthesis was found to have more of an effect on virulence in orally inoculated T. ni than S. exigua and that virulence was dependent on two factors: the ability of the host to slough occlusion-derived virus (ODV)-infected midgut cells and the rapidity with which BV was transmitted to the tracheal cells. In both host species, insects inoculated orally with the control virus transmitted BV to tracheal cells hours before those inoculated with the gp64 temporal mutant. Moreover, with early GP64 synthesis, the lag between the onset of viral gene expression in midgut and tracheal cells was only 3-4 h, supporting the conclusion that in these insects, the first systemic infections arose from ODV-derived nucleocapsids repackaged as BV. These results provide further empirical proof that early GP64 synthesis is a component of a unique and selectively advantageous baculovirus infection strategy for exploiting larval lepidopterans by counteracting developmental resistance.

Early synthesis of budded virus envelope fusion protein GP64 enhances Autographa californica multicapsid nucleopolyhedrovirus virulence in orally infected Heliothis virescens.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the type species of the Nucleopolyhedrovirus genus (Baculoviridae family), has two highly unusual traits shared by several baculovirus species. First, the occlusion-derived virus (ODV) that establishes primary infection in the midgut following its ingestion by host larvae contains multiple nucleocapsids, all of which enter the same midgut cell. Second, GP64, the envelope fusion protein of the budded virus (BV) that spreads infection beyond the midgut, is synthesized both early and late during infection. We tested the hypothesis that, together, these two traits enable parental ODV nucleocapsids to bud from infected midgut cells, essentially as BV, to establish secondary infections prior to completion of viral replication within the midgut. This "pass-through" strategy would enable the virus to counter the host's principal defense, sloughing of infected midgut cells, by accelerating the onset of systemic infections. To test this hypothesis, we created an AcMNPV recombinant, AcLate21/20-64HB, that can express gp64 only during the late phase of infection (coincident with the other structural proteins). We then compared the virulence of this virus to that of a control recombinant virus that expresses gp64 in a wild-type manner. We found that when administered orally, the control virus was far more virulent and established secondary infection earlier than AcLate21/20-64HB, but when administered intrahemocoelically, infectivity and virulence of the two recombinants were identical. Our results demonstrate that early gp64 expression is a key component of a unique and highly adaptive baculovirus infection strategy.

Comparative pathogenesis of Helicoverpa zea S nucleopolyhedrovirus in noctuid larvae.

We used a recombinant of Helicoverpa zea S nucleopolyhedrovirus containing the hsp70/lacZ reporter cassette (HzSNPV-hsp70/lacZ) to quantify mortality relationships and to elucidate early pathogenesis in two permissive hosts, Heliothis virescens and Helicoverpa zea, and one semi-permissive host, Trichoplusia ni. Fourth instar T. ni were highly resistant to fatal infection both by oral injection of occlusions and by intrahaemocoelic injection of budded virus, indicating the presence of both midgut and systemic mechanisms of resistance. In bioassays, newly moulted (4(0)) H. zea were significantly more susceptible than 4(0) H. virescens to fatal infection, but mortality levels were the same for larval cohorts inoculated 16 h after the moult (4(16)). Developmental resistance was stronger in H. zea and in both hosts, partially reversed by administration of the optical brightener M2R. In both species, developmental resistance was correlated with a reduced ability of HzSNPV to establish and/or maintain primary midgut infections. In time-course experiments using a dosage of 15 occlusions ( approximately LD(90)), lacZ expression marking the onset of primary and secondary infection was first observed in midgut columnar and tracheal cells at 4 and 12 h, respectively. Inoculation of 4(0) larvae resulted in approximately twofold more foci in H. zea larvae than in H. virescens, but H. zea larvae sloughed infected midgut cells at a faster rate. For both heliothines, interaction of occlusion-derived virus with primary cellular targets within the midgut epithelium was critical to the outcome of infection and a key process underlying acquisition of developmental resistance.

Midgut-based resistance of Heliothis virescens to baculovirus infection mediated by phytochemicals in cotton

The decrease in susceptibility to polyhedrosis disease when Heliothis virescens larvae feed on cotton is profound, limiting the utility of baculoviruses for controlling noctuids on this important crop. We observed that the mortalities of H. virescens larvae challenged with a reporter-gene construct of Autographa californica M nucleopolyhedrovirus (AcMNPV- hsp70/lacZ) and fed either lettuce or artificial diet were ≈2.5-fold higher than that of cotton-fed insects. This decrease in susceptibility on cotton was observed following oral but not intrahemocoelic inoculation of virus, and it was negatively correlated with levels of foliar peroxidase. The rates of development of both infected and uninfected larvae also were correlated negatively with levels of foliar peroxidase, and hence, were significantly lower for insects fed cotton. When Calcofluor White M2R, an optical brightener reported to enhance the retention of AcMNPV-infected midgut cells, was included in inoculum administered orally to larvae, mortality levels were equivalent regardless of diet. These results suggest that sloughing of infected midgut cells occurred at a higher rate in insects that fed on cotton compared to the other two diets, and that midgut cell sloughing is the mechanism whereby susceptibility to mortal infection by AcMNPV- hsp70/lacZ is decreased on cotton. This conclusion is consistent with previous reports that ingestion of cotton can generate reactive oxygen species within the midgut lumen that may damage midgut epithelial cells. As far as we know, this is the first study to link resistance intrinsic to the physiology of the insect (e.g., developmental resistance) and resistance conferred by host plant chemistry to a single mechanism, i.e., midgut cell sloughing.

Developmental Resistance in Fourth Instar Trichoplusia ni Orally Inoculated with Autographa californica M Nuclear Polyhedrosis Virus

Larvae of lepidopteran insects commonly become increasingly resistant to baculovirus infections as they age. The mechanism responsible for this developmental resistance is not known, but the phenomenon does not occur if the viral inoculum is administered intrahemacoelically instead of orally, which is the natural route of infection. This observation indicates that the factors mediating developmental resistance are operative during infection of the primacy target tissue, the larval midgut, and not during subsequent systemic infection. To learn more about the mechanism of developmental resistance, we orally inoculated four cohorts of fourth instar Trichoplusia ni larvae with a recombinant of Autographa californica M nuclear polyhedrosis virus expressing a reporter gene. While these cohorts differed only by a few hours in age, we found increasing resistance to infection in successively older cohorts. By assessing the presence and location of infected cells at intervals during the first 48 hr after inoculation, we identified two key factors relevant to the resistance pattern among the developmental cohorts. These factors were: (i) an age-dependent rate of establishing and/or sloughing infected midgut cells and (ii) the ability of fourth instar T. ni to completely clear infection of the midgut epithelium by ecdysis to the fifth instar.

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm, Bombyx mori, reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.

Sublethal virus infection depresses cytochrome P-450 in an insect

Insect-specific cytoplasmic polyhedrosis virus infections, endemic in many species of insects, cause the gut tissue to assume on opaque, milky-white appearance through virus multiplication and formation of polyhedral protein inclusion bodies. Electron microscopy shows that the endoplasmic reticulum membrane is severely reduced and fragmented in infected midgut cells. Metabolism of foreign, lipophilic compounds, catalyzed by the membrane-bound cytochrome P-450, is significantly depressed, and resistance to insecticides disappears. In the absence of toxicants, most insects in this condition survive with sowewhat impaired fitness.

A variant of La Crosse virus attenuated for Aedes triseriatus mosquitoes.

A plaque-purified variant was cloned from prototype La Crosse (LAC) virus. This variant (PP-31) was lethal to suckling mice by the intracerebral route, produced "wild-type" plaques in Vero and BHK-21 cells, and grew to high titers (greater than 10(7) PFU/ml) in suckling mice and in cell culture. The variant was able to orally infect the vector, Aedes triseriatus; however, it was unable to escape infected midgut cells and disseminate to secondary target organs. Large, atypical, focal accumulations of viral antigen were detected in these midguts by immunofluorescence. Orally infected mosquitoes were unable to transmit virus by bite to suckling mice or vertically to their progeny. Even after inoculation of the variant virus into mosquitoes, there appeared to be a restriction on cell to cell virus movement. The role such variants may play in the modulation of infection in an arthropod vector is discussed.

Preferential separation of cytoplasmic-polyhedrosis virus (CPV) RNAs from infected midgut cells

Viral progeny, double-stranded RNA (dsRNA), and virus-specific single-stranded RNA (ssRNA) synthesized in midgut cells of Malacosoma disstria larvae infected with cytoplasmic-polyhedrosis virus (CPV), have been separated using Mg 2+ for RNA aggregation, and lithium chloride for differential solubilization between the ds- and ssRNA. Under high concentrations of Mg 2+ (5 × 10 −3 and 10 −2 m MgSO 4), dsRNA was not affected, remaining positional at a sedimentation rate of 15S, but virus-specific ssRNA and host ribosomal RNA as single-stranded (rRNA), were separated from dsRNA by aggregation. After RNase treatment there was no appreciable contamination of virus specific ssRNA and rRNA. It was found that ds and ssRNA could be separated in 2 mLiCl on the basis of their different solubilities. The ssRNA fraction obtained by such a procedure contained only 6% dsRNA, and the dsRNA fraction was free of ssRNA. Further separation of dsRNA by both the Mg 2+ and LiCl treatments was accomplished through prolonged centrifugation which yielded 12 and 15 S components; these are the same characteristics found for viral genome RNA appearing in the sedimentation profile.

The inflammatory response of the tobacco hornworm, Manduca sexta, to infection by the microsporidian, Nosema sphingidis

A histological description is presented of the reaction of hemocytes of the tobacco hornworm, Manduca sexta, to infection by the microsporidian Nosema sphingidis. Nodule formation on the posterior portion of the midgut epithelium was detected within 24 hr post-exposure to spores. By 120 hr, the inflammatory response was striking, with extensive nodule formation at the site of infected midgut cells labial glands, Malpighian tubules, and adipose tissue. In the late stages of infection, the posterior portion of the midgut was extensively deranged, with intense hemocytic infiltration and melanization. Since few similar observations have been reported, a discussion of immune responses of insects to microsporidian infection is presented.

Occluded and free virions in midgut cells of Malacosoma disstria infected with cytoplasmic-polyhedrosis virus (CPV)

The proportion of occluded virions and free virions in infected midgut cells of Malacosoma disstria was determined at advanced stages (10 days) of cytoplasmic-polyhedrosis virus infection. In infected cells, less than 30% of the virions were occluded within polyhedral bodies, and more than 70% were found as free forms. In virus with uridine- 3H, it was found that free virions were labeled more quickly than those occluded in polyhedra. Occlusion of virions within the protein matrix of the polyhedral inclusion body did not occur until 5–10 hr after the appearance of free virions.

Multiplication of a granulosis virus in larval midgut cells of Trichoplusia ni and possible pathways of invasion into the hemocoel

The ultrastructure of the midgut epithelium of the fourth-instar cabbage looper larva of Trichoplusia ni has been described. The epithelium consists of columnar and goblet cells. A granulosis virus of T. ni infects and multiplies in columnar midgut cells and in fat cells. The goblet cells appear not to be infected. Complete virus particles with developmental (or outer) membranes have been found, 24 hours after virus ingestion, in some of the intercellular spaces between midgut cells. There is evidence of virus multiplication in the nuclei of columnar cells as early as 24 hours after virus ingestion. A few virus particles occur in the cytoplasm near the bases of the microvilli, but most are found in the basal portion of the cytoplasm of the infected cells. Some particles also appear in the intercellular spaces at the bases of the columnar cells and in the basement membrane. The infected midgut cells contain “naked” and complete virus particles, but very rarely virus inclusion bodies (capsules). Occasionally hexagonal inclusions occur in virus-infected cells, but they do not seem to have virus particles in them. Based on these observations, we have postulated two pathways by which the ingested granulosis virus may invade into the hemococl of the cabbage looper larva.

Site of viral RNA synthesis within the midgut cells of the silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

The pattern of nucleic acid synthetic activity within the midgut cells of healthy larvae of the silkworm, Bombyx mori, and of larvae infected with the cytoplasmic-polyhedrosis virus was demonstrated by means of autoradiography with labeled nucleic acid precursors. Five hours after injection of uridine-H 3, the healthy cells generally incorporated the labeled material into cytoplasmic RNA and partly into nucleic RNA, whereas the diseased cells on the 2nd to the 5th day after virus inoculation incorporated much of the labeled uridine into nucleic RNA and some into cytoplasmic RNA. In the nuclei of virus-infected cells, the nucleic label appeared most densely over the nucleoli. Thus the distinct difference in the uptake of the RNA precursor between the healthy and infected cells indicated that the nucleolus of the infected cell may be a site of the viral RNA synthesis. Autoradiograms with thymidine-H 3 revealed no essential difference in the pattern of DNA synthesis between healthy and diseased midguts, and only a few cells incorporated the labeled material into their nuclei. At the late stage of virus infection, however, when some infected midgut cells eventually degenerated, there was a slight increase in the nucleic label in the newly generated cells.