Persistence of insect viruses in field populations of alfalfa insects

The persistence of viruses of five insects was observed in alfalfa fields. The insects were Autographa californica, Colias eurytheme, Pseudaletia unipuncta, Spodoptera exigua, and Trichoplusia ni. The isolated viruses were the granulosis (GV), the cytoplasmic-polyhedrosis (CPV), and the nuclear-polyhedrosis (NPV) viruses. The viruses persisted in the soil, on the alfalfa foliage, and in alternate hosts. In the soil, the viruses persisted even during the winter months when no foliage remained on the plants. Alfalfa sprouts harboring virus-infected larvae of C. eurytheme and S. exigua produced virus infections in larvae of these insects, but those with larvae of A. californica and P. unipuncta did not cause virus infection. The GVs and CPVs isolated from these insects were transmitted to nearly all of the other four species, but the NPVs appeared to be host specific.     

Specificity of receptor sites on insect cells for the synergistic factor of an insect baculovirus

The granulosis virus of the armyworm, Pseudaletia unipuncta, contains a synergistic factor (SF) which enhances the in vitro and in vivo infections of baculoviruses. The SF agglutinates certain insect cells but not the cell line of Trichoplusia ni (TN368). A single application of the SF to TN368 cells causes an interference of the infection of a nuclear polyhedrosis virus of Trichoplusia ni (TnMNPV). The interference increases with higher concentrations of the SF. Multiple applications of the SF result in a decrease in interference and possibly even in an enhancement. These observations suggest that the receptor sites on the cell plasma membrane of TN368 cells are not the same for the SF and for the virus.     

Phospholipid,an enhancing component in the synergistic factor of a granulosis virus of the armyworm,Pseudaletia unipuncta

The synergistic factor (SF) in the capsule of a granulosis virus (Hawaiian strain) of the armyworm, Pseudaletia unipuncta, contained polypeptides and phospholipids. Its molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 126,000 ± 8,700. The capsule proteins were digested by a proteinase released from the capsule under alkaline conditions, and by trypsin added to the proteinase-free capsules. Neither enzyme affected the synergistic factor or its activity. The synergistic factor was slowly depolymerized by 2% sodium dodecyl sulfate and was more rapidly depolymerized when phospholipase C (phosphatidylcholine cholinephosphohydrolase) was also added. Phospholipase C alone did not decompose the synergistic factor, but it did destroy the capacity of the synergistic factor to enhance the nuclear polyhedrosis virus. In contrast, phospholipase A₂ (phosphatidyl 2-acylhydrolase) had no effect on the synergistic factor. The different reactions of the two phospholipases on the synergistic factor suggested that the hydrophilic group of the phospholipid was exposed to the action of phospholipase C and was associated with the synergistic activity. This interpretation was supported by the detection of a phospholipid in the SF by thin-layer chromatography.     

Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SF), which is present in the capsule matrix protein of a granulosis virus of the armyworm, Pseudaletia unipuncta, enhances baculovirus infection in armyworm larvae. The site of action of the SF was investigated. The oral inoculation of SF did not enhance the infectious hemolymph virions which had been inoculated into the hemocoel. The SF also did not enhance the infection of purified enveloped virions when both virus and SF were inoculated into the hemocoel, but enhancement occurred when they were inoculated orally. Thus, the activity of the SF was confined to the midgut lumen. Observations with ferritin-conjugated antibody indicated that the site of action of SF was the cell membrane of the microvillus. There were more ferritin particles attached to midgut cell membranes of larvae inoculated orally with SF than to those of control larvae inoculated with buffer.     

Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with ¹²⁵I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures.     

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.     

Protein components of two strains of granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera,Noctuidae)

A synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain of a granulosis virus (GV) infect the armyworm, Pseudaletia unipuncta. The protein components of the enveloped virions and of the capsule (inclusion body) were compared between the two strains. When the enveloped virions of both strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein patterns were similar except for minor peaks in the higher molecular weight region. On the other hand, the capsule proteins differed between the two strains when examined with immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. The capsule proteins of GVH were composed of two major proteins, one a structural protein and the other the protein of the synergistic factor. The capsule protein of GVO, however, had only the structural protein. The rocket immunoelectrophoresis and affinity chromatography indicated that the structural proteins of the two strains were partially dissimilar. The molecular weight of the structural protein of GVO was 29,100 ± 500 and that of GVH was 28,700 ± 500. The amount of synergistic factor in a GVH capsule was about 5% of the dissolved capsule components.     

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.     

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.     

Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Occurrence and accumulation of viruses of Trichoplusia ni in treated field plots

Concentrations of the nuclear-polyhedrous virus (T. ni NPV) and the granulosis virus (T. ni GV) of the cabbage looper, Trichoplusia ni, in soil and on foliage were monitored up to 4 years after treatment.A single application of T. ni NPV to soil in August or 5 foliar applications of the virus at 10-day intervals in August and early September maintained substantial concentrations of the virus on foliage and high concentrations of the virus accumulated in soil. With development of natural epizootics of the virus disease in populations of the host larvae in September and October, substantial concentrations of the virus accumulated in soil and on foliage in nontreated plots, eventually becoming equal in amount with the virus in virus-treated plots. The virus accumulated more slowly in plots treated with chemical insecticides or Bacillus thuringiensis because few host larvae survived to support late-season epizootics of the disease. Small quantities of T. ni NPV were detected in heads of cabbage harvested from the plots in October.Long-term studies in which nontreated plots and plots treated with T. ni NPV or T. ni GV were replanted for up to 4 years after treatment showed that concentrations of T. ni NPV in surface soil remained constant during the winter but were reduced by dilution during cultivation preparatory to planting in the spring. T. ni NPV accumulated during the late summer and autumn with development of epizootics of the disease in populations of host larvae. Increased concentrations of the virus in soil coincided with increased concentrations on leaves in each year. T. ni GV did not persist on leaves or in soil following application and only small amounts were found 2 years after application.T. ni NPV disease was prevalent in September and October in populations of host larvae in plots in which substantial residues of the virus were found. These epizootics contributed substantially to late-season control of the looper after completion of spraying.     

粘虫颗粒体病毒增效因子的基因定位

参考粉纹夜蛾Trichoplusia ni 颗粒体病毒增强因子的基因序列,设计PCR引物,用PCR反应扩增出特异性产物。用EcoRⅠ、BamHⅠ双酶酶切处理PCR反应产物,然后克隆到质粒pUC19中,构建重组质粒pUC19-SF;对重组质粒pUC19-SF中的外源片段测序,结果证明PCR扩增产物是粘虫颗粒体病毒PuGV-Ps增效因子基因的一段序列。重组质粒pUC19-SF的插入片段标记为探针,通过Southern杂交将增效因子基因定位于PuGV-Ps病毒基因组的多种酶切片段上。     

Biochemical properties of viral envelopes of insect baculoviruses and their role in infectivity

The enveloped virions of a nuclear polyhedrosis virus (NPV) and those of a granulosis virus (GV) of the armyworm, Pseudaletia unipuncta, were isolated and purified from their inclusion bodies. The enveloped virion of NPV contained a large amount of phosphatidyl choline which was not detected in that of GV. The total electric charges distributed on the surface of the envelopes of NPV and GV were negative in neutral and alkaline solutions. Although there was little difference in charges between NPV and GV, the charge was less negative in NPV than in GV. When the negative charges were neutralized by cationic detergents, the NPV infectivity was enhanced.     

Differential susceptibility of lepidopterous pupae to infection by the nematode Neoaplectana carpocapsae

Prepupae of Galleria mellonella, Spodoptera exigua, and Pseudaletia unipuncta were highly susceptible to infection by the nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus. On the other hand, pupae of the three species were significantly different in their susceptibility to the nematode. G. mellonella pupae were highly susceptible (100% mortality), S. exigua pupae were moderately susceptible (ca. 75% mortality), and P. unipuncta pupae were least susceptible (ca. 54% mortality). In the latter two species, many pupae died without any nematode development, but the typical signs associated with a nematode infection were evident. Age of the pupae and increasing the dosage of the nematode did not significantly affect mortality of S. exigua or P. unipuncta.     

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.     

Isolation and characterization of a synergistic enzyme from the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor than enhances the infection of a nuclear polyhedrosis virus in the armyworm, Pseudaletia unipuncta, was isolated from the occlusion body (capsule) of a granulosis virus of the armyworm. Disc electrophoresis indicated that the purified factor was a single homogeneous compound. Chemical identification and amino acid analysis showed that it was a simple protein, with a molecular weight of 152,000–163,000. Proteolytic enzymes did not markedly reduce the activity of the factor. It could be stored at ?20°C or lyophilized. The synergistic factor displayed properties of an enzyme. It enhanced the hydrolysis of p-nitrophenyl esters of fatty acids with an optimum of pH 9.0. The relative hydrolytic activity increased with increase in number of carbon atoms in the fatty-acid chain from 2 to 8 and gradually decreased with the number of carbon atoms from 10 to 18. Copper sulfate markedly and mercuric chloride completely prevented enhancement of the hydrolysis of butyrate. When the synergistic factor was fed to larvae with mercuric chloride, it did not enhance the nuclear polyhedrosis virus.     

Susceptibility of early larval stages of Pseudaletia unipuncta and Spodoptera exigua (Lepidoptera: Noctuidae) to the entomogenous nematode Steinernema feltiae (Rhabditida: Steinernematidae)

Early stages (neonate to 7- or 8-day-old larvae) of Spodoptera exigua and Pseudaletia unipuncta were exposed to the entomogenous nematode, Steinernema feltiae, at concentrations of 0, 10, 25, 60, 100, or 200 nematodes per larva. Larvae of both species were susceptible to nematode infections. However, neonate larvae of S. exigua were significantly less susceptible to nematode infection than 3- or 8-day-old larvae at or above 50 nematodes per larva. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 68 to 74% while mortalities of 3- and 8-day-old larvae ranged from 91 to 100%. The results with P. unipuncta showed similar trends as described for S. exigua, albeit at a lower mortality level and usually with no statistical differences. Mortalities of neonate larvae exposed to 50 or more nematodes ranged from 34 to 44% while mortalities of 7-day-old larvae ranged from 32 to 91%.     

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.     

In Vivo and In Vitro Analysis of Baculovirus ie-2 Mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.