Robert R. Granados

In vivo pathway of Autographa californica baculovirus invasion and infection.

The pathway of Autographa californica nuclear polyhedrosis virus (AcNPV) infection in cabbage looper, Trichoplusia ni, larval midgut cells was studied by ultrastructural and virus titration methods. Enveloped virions interacted with microvilli of columnar cells resulting in apparent fusion of the viral envelope and microvillus membrane. After entry into the cell cytoplasm, the intact nucleocapsids appeared to enter the nucleus through nuclear pores, and uncoating of the viral genome took place in the nucleoplasm. Viral progeny were first observed at 8 hr postinoculation (p. i.) and the developmental cycle of the virus was essentially completed by 24 hr p.i. Inoculum virus nucleocapsids also moved to the basal plasma membrane and budded into the hemocoel through the basal lamina within 0.5 hr p.i. We propose that this budded virus, possessing an envelope with a peplomer structure, is the primary inoculum for the systemic invasion of the insect host.

Calcofluor disrupts the midgut defense system in insects.

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.

A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus

The replication of Trichoplusia ni SNPV (TnSNPV) in a new Lepidopteran cell line, BTI-Tn-5B1-4 (Tn-5B1-4), is presented. This cell line was highly susceptible to TnSNPV with infection rates exceeding 90% by 48 hr postinfection (p1). Budded virus (BV) was present in the culture medium 14 hr pi and reached a maximum titer of 3.2 × 10 6 TCID 50 /ml 48 hr pi. Cell-associated virus was produced 12 hr pi and reached a maximum titer of 2.4 × 10 6 TCID 50 /ml 36 hr pi. Serial passage of TnSNPV was initiated in Tn-5B1-4 cells with infectious hemolymph obtained from T. ni larvae infected with TnSNPV occlusion bodies (OBs). TnSNPV was subsequently grown in this cell line for 60 serial passages. Levels of BV infectivity were maintained at approximately 3.2 × 10 5 TCID 50 /ml through passage 60. Early passage virus (i.e., 1–7 passages) resulted in approximately 500 to 700 OB/cell, whereas later passages (i.e., 7–60) showed an average decrease from 450 to 100 OB/cell. Bioassay results showed high infectivity (approximately 95%) with early passage virus; however, infectivity dropped off rapidly in later passages with no larval infectivity seen after passage 20. In addition, the effects of cryopreservation on virulence and yield of TnSNPV-BV, produced in the Tn-5B1-4 parental cell line, BTI-Tn-5B1, at 4 and -100°C, were investigated. By 6 weeks, BV titers for samples stored at both temperatures had decreased by as much as one order of magnitude. Although gradual decreases in titer were observed under both storage conditions, −100°C appeared to be more suitable for long term cryopreservation.

Alteration of a lepidopteran peritrophic membrane by baculoviruses and enhancement of viral infectivity

The peritrophic membrane (PM), which lines the midgut of many insect species, has several functions. In particular, it may serve as a mechanical barrier to invading microorganisms. The protein composition of the PM from healthy and baculovirus-treated Trichoplusia ni (cabbage looper) larvae was analyzed by polyacrylamide gel electrophoresis. A specific interaction took place between baculoviruses and the PM of susceptible T. ni larvae. A 68-kDa glycoprotein of the PM disappeared within 15 min postinoculation with occlusion bodies of either Autographa californica nuclear polyhedrosis virus (AcMNPV) or T. ni nuclear polyhedrosis virus (TnSNPV). In contrast, inoculation of larvae with a T. ni granulosis virus (TnGV) resulted in the disappearance of three distinct major glycoproteins with molecular weights of 253, 194, and 123 kDa. PMs of virus-treated larvae were very fragile compared with those of untreated controls, indicative of a physical/chemical change in their structure. T. ni larval bioassays showed that a factor, present in the TnGV granulin or AcMNPV polyhedrin, enhanced the infectivity of AcMNPV. These data showed that a factor present in the occlusion bodies of three distinct baculoviruses can cause specific biochemical and structural changes in the PM. The biological significance of these observations in relation to increased larval infection is not known at this time.

COMPARATIVE RECOMBINANT PROTEIN PRODUCTION OF EIGHT INSECT CELL LINES

SummaryA recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.

Molecular Cloning and Sequencing of a Novel Invertebrate Intestinal Mucin cDNA

The first invertebrate intestinal mucin, termed insect intestinal mucin (IIM), was recently identified fromTrichoplusia ni larvae (Wang, P., and Granados, R. R. (1997) Proc. Natl. Acad. Sci. U. S. A., in press). We report the cDNA cloning and sequencing of IIM, which is only the second completely sequenced intestinal mucin after human intestinal mucin, MUC2. To clone and sequence the cDNA for IIM, a T. ni larval midgut cDNA expression library was constructed and screened with an anti-IIM antiserum. Two full-length cDNA clones for IIM were identified and sequenced. The deduced proteins from the two cDNA clones contained 807 and 788 amino acid residues, respectively. The structural organization of IIM is similar to that of MUC2, containing a 25-amino acid signal leading sequence and two threonine/proline/alanine-rich tandem repeat domains flanked by cysteine-rich sequences. One tandem repeat domain contained two repeating units, TTTQAP and AATTP, and the other contained one repeating unit, TAAP. The cysteine-rich regions showed potential chitin binding features. By immunolocalization in tissue sections, it was determined that IIM is expressed in midgut tissues. The IIM mRNA is abundant in the midgut tissue, and Northern blot analysis indicated that IIM transcripts were not polydispersed as is found in mammalian mucin transcription.

Mycoplasma diseases of plants and insects.

Publisher Summary This chapter explains the Mycoplasma diseases of plants and insects. It discusses the nomenclature and classification of the order Mycoplasmatales. The nonviral nature of the numerous graft-transmissible, arthropod-borne disease agents of plants is established. Electron microscopy reveals the characteristic morphology of the presumptive micro-organisms observed in diseased phloem cells and in various vectors, but absent from healthy phloem or from non-infected vectors. Plants can recover from yellows-type diseases when treated with sulfa drugs that definitely do not affect mycoplasmas, and they sometimes recover when treated solely with fertilizers. The chapter discusses on only specific criteria, accepted by microbiologists, should be used in the determination of the actual etiology of the presumptive mycoplasma diseases. These criteria require the cultivation of the isolated agents on solid media with the production of mycoplasma colonies, the reinoculation of susceptible healthy plants with the cultured mycoplasmas, and the subsequent reproduction of the original disease.

General analysis of receptor-mediated viral attachment to cell surfaces.

Viruses are multivalent particles that attach to cells through one or more bonds between viral attachment proteins (VAP) and specific cellular receptors. Three modes of virus binding are presented that can explain the diversity in binding data observed among viruses. They are based on multivalency of attachment and spatial versus receptor saturation effects which are easily distinguished based upon simple criteria. Mode 1 involves only monovalent virus/receptor binding. Modes 2 and 3 involve multivalent bonds between the virus and cell; however, in mode 3 space on the cell surface becomes saturated before receptors. A model is developed for viral attachment that accounts for nonspecific binding, receptor/virus interactions, and spatial saturation effects. The model can describe each mode in different limits and can be applied to virus binding data to extract key physical information such as receptor number and affinity. These values are used to postulate the type of VAP/receptor interaction involved and to predict binding at different parameter values. For the mode 2 binding of Adenovirus 2, the model predicts a receptor number of 4-15 x 10(3) on HeLa cells and an affinity of 2-6 x 10(7) M-1 which closely approximate experimental estimates. For the binding of three, broad-host-range, enveloped viruses, Semliki Forest virus, Vesicular Stomatitis virus, and the baculovirus, Autographa californica nuclear polyhedrosis virus, the model predicts receptor numbers of 10(5) or greater and affinities in the range of 10(4) to 10(5) M-1. These values are indicative of a VAP/oligosaccharide interaction which has been documented for a number of other viruses. Experimental evidence is presented that is the first to demonstrate that baculovirus binding is mediated by a cell surface receptor.

Early events in the infection of Heliothis zea midgut cells by a baculovirus

Abstract Virus adsorption, penetration and uncoating were observed in midgut cells of laboratoryreared larvae of Heliothis zea (cotton bollworm) fed per os with the H. zea nuclear polyhedrosis virus. Within 2 hr post-inoculation (p.i.), enveloped virions interacted with microvilli of columnar cells resulting in the apparent fusion of the viral envelope and microvillus membrane. The viral envelope was lost during the entry process inasmuch as only nonenveloped nucleocapsids were observed within microvilli. After entry into the cell cytoplasm, the intact nucleocapsids appeared to gain access into the nucleoplasm by interacting end-on with nuclear pores. Within 2 to 4 hr post-inoculation, numerous nucleocapsids could be seen in the nucleoplasm, a number of which appeared to be empty or partially empty. These findings show that uncoating of this virus took place within the nucleus and not at the site of the nuclear pores as suggested for some baculoviruses.

Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection

SummaryDefective interfering particles, present within a high passage inoculum ofAutographa californica nuclear polyhedrosis virus (AcMNPV), interfered with recombinant β-galactosidase and infectious virus production in three insect cell lines. These particles were Selected during serial passage, were missing large parts of the AcMNPV genome, and caused large reductions in β-galactosidase production at multiplicities of infection above 0.01 pfu/cell.