Serological relationships among plusiinae baculoviruses

Antisera were produced against nucleocapsids, NP-40 detergent soluble proteins, or polyhedral protein of the multiply embedded nuclear polyhedrosis virus (MNPV) of Autographa californica, nucleocapsids of Trichoplusia ni singly embedded virus (SNPV), and polyhedral protein of Lymantria dispar MNPV. Antigens consisting of nucleocapsids, NP-40 soluble proteins, and polyhedral protein were prepared from A. californica MNPV, T. ni MNPV, L. dispar MNPV, Rachiplusia ou MNPV, T. ni SNPV, and Pseudoplusia includens SNPV. Radial immunodiffusion patterns formed with Plusiinae nucleocapsid antigens and antiserum to nucleocapsids of A. californica MNPV or T. ni SNPV revealed a distinction between multiply and singly embedded viruses. The same alignment of Plusiinae viruses was observed in reactions between A. californica NP-40 soluble protein antiserum and the NP-40 soluble protein fractions from the Plusiinae NPVs. There were no reactions between the Plusiinae SNPV nucleocapsid antigens and the A. californica MNPV nucleocapsid antiserum. However, there were faint precipitin bands between MNPV nucleocapsid antigens and T. ni SNPV nucleocapsid antiserum. Each of the polyhedral protein fractions from the Plusiinae formed a single precipitin band with the antiserum to polyhedral protein of either A. californica or L. dispar. The precipitin bands formed with the A. californica antiserum by polyhedral proteins of T. ni SNPV, P. includens SNPV, and R. ou MNPV were confluent, and shared partial identity with those formed by A. californica MNPV and T. ni MNPV. All precipitin bands formed by Plusiinae polyhedral proteins against antiserum to L. dispar polyhedral protein were confluent, and shared partial identity with that formed by L. dispar polyhedral protein.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

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 of M. brassicae. Both viruses produced a greater yield in M. 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.     

Infectivity of a nuclear polyhedrosis virus from the alfalfa looper,Autographa californica,after passage through alternate hosts

A nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica, was found to infect several species of caterpillars including the cabbage looper, Trichoplusia ni; the beet armyworm, Spodoptera exigua; and the saltmarsh caterpillar, Estigmene acrea. Studies were therefore conducted to determine the quantitative effects of passage through the alternate hosts, S. exigua and E. acrea, on the infectivity of this virus to newly hatched first-instar cabbage looper larvae. When 11 preparations of polyhedra obtained from a like number of primary passages through the original or alternate hosts were assayed and the mortality at 7-, 10-, and 14-day intervals were subjected to probit analysis, the LD₅₀s for the three intervals differed but those for the preparations at any given interval did not. Therefore, any of the three hosts could be used to propagate the virus, and whichever proves the easiest to rear and provides the highest yields of polyhedra can be selected.     

Measurement of Surface Charge of Baculovirus Polyhedra

The isoelectric points of three baculoviruses, Trichoplusia ni nuclear polyhedrosis virus (NPV), T. ni granulosis virus, and Spodoptera littoralis NPV were identified by cell electrophoresis. At neutral pH polyhedra were negatively charged. T. ni NPV polyhedra were reacted with a number of reagents which could potentially attach to or degrade their surface structure. This gave information on the components that contribute to the charge profile of T. ni NPV. This is discussed in relation to the use of polyhedra as biological control agents against insect pests.     

Infectivity of nuclear polyhedrosis virus extracted with digestive juices

Autographa californica NPV, which had been obtained by dissolving polyhedra in the digestive juice of Estigmene acrea larvae, was infectious to a Trichoplusia ni cell line (TN-368). Virions thus botained were infective, and as few as 0.0025–0.005 polyhedral equivalents could infect newly transferred tissue culture cells. Activity decreased after 8 min of digestion.     

Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, produced in cells grown in vitro

A virus isolated from the alfalfa looper, Autographa californica, replicated successfully and rapidly in a suspended ovarian cell line of the cabbage looper, Trichoplusia ni. Polyhedra were observed in the nucleus of cells within 20 hr after inoculation. The cytopathological changes typical of nuclear polyhedrosis infections were observed, and an average of 64 polyhedra/cell were produced. These polyhedra were quantitatively as infectious to cabbage looper larvae as those produced in vivo. In addition, they were infective to Heliothis virescens, Pectinophora gossypiella, Spodoptera exigua, A. californica, and Anagrapha falcifera.     

Comparative recombinant protein production of eight insect cell lines

Summary A 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.     

Relative susceptibility of several noctuid species to a nuclear polyhedrosis virus from Heliothis armiger

Several alternate hosts were tested for their relative susceptibility to an isolate of Galleria mellonella nuclear polyhedrosis virus. Neonate Trichoplusia ni, Heliothis zea, and Manduca sexta were all susceptible to per oral administration of purified polyhedra. Of the three alternate species tested, T. ni was the most susceptible, and exhibited the most variable mortality response over the dose range tested, while M. sexta was the least susceptible. We believe this represents the first report of a lethal virus infection in a sphingid species, and useful parameters for the successful inoculation of alternate hosts are discussed.     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.     

Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus

The TN-368 tissue culture line of the cabbage looper, Trichoplusia ni, has been cloned. The doubling times of three clones at 27°C were 27.6 ± 3.4 hr, 21.9 ± 1.7 hr, and 27.4 ± 5.9 hr and that of the uncloned culture was 15.8 ± 1.5 hr. Growth of cells in all cultures was arrested after infection with a nuclear polyhedrosis virus of T. ni. There was little difference in the yield of polyhedra from cultures of uncloned or cloned cells infected at a multiplicity of infection (m.o.i) = 4. Yields of polyhedra were about the same when a m.o.i. was in the range of 0.01–4.0, but the yield tripled in the range m.o.i. = 20–30. At higher multiplicities, up to m.o.i. = 500 the yield of polyhedra progressively fell. It is concluded that the observed variation in numbers of polyhedra borne by individual cells in culture is not due to genetic variability among cells, nor can it be accounted for as a consequence of differing m.o.i. by virus. It is postulated that variation in polyhedra yield among cells in culture may be due to such factors as (1) strain differences in the virus, (2) the stage in the cell cycle at which a particular cell is present when infected.     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Propagation of Autographa californica nuclear polyhedrosis virus in cell culture: Methods for infecting cells

Autographa californica nuclear polyhedrosis virus (AcNPV) produced in Trichoplusia ni (TN-368) cells was used to infect other cell cultures. Methods were developed to recover and obtain high titers of virus from infected cells for subsequent use as inocula. To release cell-associated nucleocapsids, the cells were lysed by sonication and freeze-thawing. The infectivity of enveloped nucleocapsids was greatly reduced by freeze-thawing, while sonication was not as detrimental. The titer of plaque-forming units (pfu) was reduced about 12-fold when passed through 0.45-μm filters. The virus and cells were manipulated to determine the most efficient methods for inoculating cells while yielding the highest numbers of polyhedra. The viral inocula may be left on cells during virus replication, and cells may be centrifuged at 380 g prior to exposure to virus without affecting the yield of polyhedra. The production of polyhedra is affected by cell density, and, of the densities tested, 7.65 × 10⁵ cells/ml yielded the maximum number of polyhedra per cell (142). However, the highest number of polyhedra per milliliter of culture (2.2 × 10⁸) was obtained with 3.8 × 10⁶ cells/ml. The numbers of polyhedra per cell did not vary when cells were taken from fermentor cultures at 0–144 hr and were infected with virus.     

Use of primary fat body cultures for the study of baculovirus replication

Fat body cultures of Trichoplusia ni and Estigmene acrea were established for use in the study of the two baculoviruses Autographa californica nuclear polyhedrosis virus (AcNPV) and Estigmene acrea granulosis virus (EaGV), respectively. Multiplication of AcNPV observed by phase and electron microscopy was correlated with an increase in viral specific proteins as determined by indirect enzyme-linked immunosorbent assay (ELISA). Although EaGV morphogenesis was not observed in fat body cultures, an increase in specific proteins of this virus could be detected with the ELISA.     

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.     

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein

Summary The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinantAutographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established fromSpodoptera frugiperda, Trichoplusia ni, andMamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in theM. brassicae-derived MB0503 cell line was Endo H resistant. The commonly usedS. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.     

The underestimated N-glycomes of lepidopteran species

BackgroundInsects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability.MethodsUsing an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line.ResultsWe detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed.ConclusionThe lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species.SignificanceThe occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.     

Serological study on the transmission of a granulosis virus of the armyworm,Pseudaletia unipuncta (Lepidoptera: Noctuidae), to other lepidopterous species

The synergistic (Hawaiian) strain of a granulosis virus (GV) from the armyworm, Pseudaletia unipuncta, was transmitted to Spodoptera exigua, Autographa californica, and Trichoplusia ni. The viruses isolated from these hosts were tested by radial double-immunodiffusion (RDD) and immunoelectrophoresis (IE) for their relationship to the original virus. Untreated and heat-treated virus inclusion bodies (capsules) were compared for their antigenic properties but no differences were detected. The antiserum elicited against the whole capsule was more sharply specific for the antigenic determinants than the one elicited against the dissolved capsule proteins. The viruses obtained from S. exigua and T. ni elicited precipitin lines that differed from those of the P. unipuncta GV in their electrophoretic mobility with the one-trough IE method; however, with the two-trough method, the lines fused indicating that the antigens were identical. The major precipitin line indicating identity of the viruses wasthat produced by the synergistic factor (SF) purified from the capsule proteins of the synergistic GV strain. The presence of SF in the GV produced in alternate hosts indicated that its production was virus directed. The SF was not detected in the GVs of Laspeyresia pomonella and Pieris rapae and in the nonsynergistic Oregonian GV of P. unipuncta. A field-collected GV from S. exigua exhibited a different precipitin pattern from that of the synergistic GV, but one of the precipitin lines shared partial relationship to the SF.     

Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses

Two media supporting the growth of several established lepidopteran cell lines in monolayer and suspension culture are described. The medium designated $\text{BML-TC}\text{10}$ was developed specifically as an inexpensive medium for production of cells of Spodoptera frugiperda and the homologous nuclear polyhedrosis virus (NPV) of this species. Simultaneously, a second medium was formulated in which the amino acid requirements were provided by enzymatic protein hydrolysates, one of which was termed $\text{BML-TC}\text{7A}$. Several cell lines could be adapted easily to this medium. $\text{BML-TC}\text{10}$ supported growth of S. frugiperda cells and production of the NPV's of S. frugiperda and Autographa californica. $\text{BML-TC}\text{7A}$ supported the growth of cells of S. frugiperda. Carpocapsa pomonella, Heliothis zea, and Trichoplusia ni. Cells of the latter produced the polyhedra of T. ni and A. californica NPV's in this medium.     

Comparative Replication of Lymantria dispar Nuclear Polyhedrosis Virus Strains in Three Continuous-Culture Cell Lines

We compared the replication of the gypsy moth (Lymantria dispar) nuclear polyhedrosis virus in two new cell lines, from embryos and fat body of L. dispar, and in a previously available ovarian cell line. Three virus isolates (the Hamden strain [LDP-67] used commercially as GYPCHEK, a plaque-purified clone of Hamden [5-7d], and an isolate from Abington, Mass. [Ab]) were each tested on the three cell lines. The fat-body-derived cell line proved best in terms of occlusion body production for all three virus strains, with the highest yield produced by the Abington strain. On the basis of these results, we conclude that a more efficient in vitro production of gypsy moth virus can be obtained by using the fat body cell line in conjunction with the Abington strain of the virus.     

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.