Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.     

USE OF SERIAL SECTIONS TO DELINEATE THE STRUCTURE OF PORTHETRIA DISPAR VIRUS IN THE ELECTRON MICROSCOPE

Consecutive serial sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus revealed bundles of viral rods scattered and oriented at random within the polyhedral body. Each bundle was entirely surrounded by a dense, sharply defined membrane. The rods measured 18 to 22 mµ in diameter and averaged 280 mµ in length. No spherical viral particles were encountered. The effects of variable compression and periodic distortion of the sections on the appearance of the virus are discussed.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

THE MACROMOLECULAR PARACRYSTALLINE LATTICE OF INSECT VIRAL POLYHEDRAL BODIES DEMONSTRATED IN ULTRATHIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Thin sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus and from silkworm larvae infected with B. mori virus revealed viral particles contained within a pseudohexagonal, macromolecular, paracrystalline lattice. The gipsy moth virus occurs in bundles of one to eight rods enclosed by a limiting membrane. The particles of the silkworm virus, although generally occurring singly, also possess a limiting membrane. The macromolecules appear to be dense, discrete particles when cross-sectioned and to form dense bands by superimposition when longitudinally or obliquely sectioned at certain angles. Calculations of macromolecular size have been made.     

Pathogenicity of a nuclear polyhedrosis virus of the almond moth, Cadra cautella

Tests were conducted with neonate Cadra cautella larvae to determine the pathogenicity of a nuclear polyhedrosis virus. A bioassay on an agar base diet showed that concentrations of 0.25, 0.50, 2.00, and 4.00 polyhedra/mm² killed 27, 55, 87, and 92% of the test larvae, respectively. A study of the time of death showed that most larvae died on the 9th or 10th day after exposure to 4 polyhedra/mm² at 27°C. When larvae were exposed to 8, 16, 32, and 64 × 10³ polyhedra/g of bran diet, recorded mortalities were 18, 22, 48, and 80%, respectively. All the samples of virus in bran diet which were incubated at various temperatures for 7, 14, and 28 days remained stable at all test conditions except the sample incubated at 42°C for 14 days, and those held at 37° and 42° for 28 days. Larvae of C. cautella, Plodia interpunctella, Ephestia elutella, and Paramyelois transitella placed on a diet with 40 × 10³ polyhedra/g had mortalities of 75, 59, 16, and 4%, respectively. Light and electron microscopical examination of P. interpunctella cadavers showed that they were infected with a multiply occluded nuclear polyhedrosis virus.     

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

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.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.     

A nonoccluded virus of the army cutworm

A nonoccluded virus was isolated from larvae of the army cutworm, Euxoa auxiliaris. Infected larvae became lethargic and shrunken, and death usually occurred 12–20 days after infection. The primary site of viral infection and replication appeared to be the nuclei of midgut epithelial cells; however, virus replication also occurred in cells of the tracheal matrix and in muscle. Nuclei in early stages of the infection contained large granular areas with the chromatin scattered near the nuclear membrane. These areas differentiated into viral particles that measured 24 nm and formed crystalline arrays, occasionally 10 μm long. Disruption of the nuclear membrane liberated these arrays of particles into the cytoplasm. Fluorescence microscopy studies indicated that the viral particles contained DNA. The crystalline arrays were Feulgen positive. The virus also infected larvae of the armyworm, Pseudaletia unipuncta, and corn carworm, Heliothis zea, in laboratory tests.     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

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.     

Aspects of the cross transmission to Galleria mellonella of a Baculovirus from the alfalfa looper, Autographa californica

The nuclear polyhedrosis virus originally isolated from the alfalfa looper, Autographa californica, was successfully transmitted to the greater wax moth, Galleria mellonella. Both the many polyhedra per nucleus (MP) and the few polyhedra per nucleus (FP) plaque variants of this virus were found to be infective when injected intracoelomically. When polyhedra of each plaque variant were fed to G. mellonella larvae, a difference in response was observed; the MP plaque variant was estimated to be 30 times more infective than the FP variant.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

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.     

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

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.     

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.