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.     

Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.     

Analysis of deoxyribonucleic acid from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Deoxyribonucleic acid (DNA) from isolates of five nuclear polyhedrosis viruses (NPV) from lepidopterous hosts of the noctuid subfamily Plusiinae was analyzed by ion-exchange and paper chromatography. Viruses and production hosts were: Trichoplusia ni singly embedded virion type (SEV) from T. ni, Pseudoplusia includens SEV from P. includens, T. ni multiply embedded virion type (MEV) from T. ni, Autographa californica MEV from A. californica, A. californica MEV from T. ni, and Rachiplusia ou MEV from R. ou. Neither uracil nor 5-methyl cytosine was detected in the DNAs. Adenine:thymine (A:T) and guanine:cytosine (G:C) ratios were nearly constant for all the NPVs. AT:GC ratios for the SEVs were 1.60 and 1.57 and were clearly separable from those of the MEVs which ranged from 1.32 to 1.38. No differences in DNA composition within SEV or MEV groups were apparent.     

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.     

Polypeptide analysis of genotypic variants of occluded Heliothis spp. baculoviruses

The structural polypeptides of 12 baculovirus isolates which included nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs) obtained from four different species of the insect genus Heliothis collected in different geographical regions of the world were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The matrix proteins were compared according to their molecular weights and peptide profiles produced after limited proteolysis. Examination of the matrix and virion polypeptide profiles revealed three major polypeptide phenotypes which corresponded to the three baculovirus morphological groups; singly embedded nuclear polyhedrosis viruses (SNPVs), multiply embedded nuclear polyhedrosis viruses (MNPVs), and granulosis viruses (GVs). Enveloped nucleocapsid polypeptide profiles of isolates within each NPV phenotype differed in only one polypeptide whereas the two GV isolates differed by as many as five polypeptides. Nucleocapsid polypeptide profiles of isolates within each of the NPV subgroups were identical while those profiles from the GV nucleocapsids differed slightly in molecular weight of one polypeptide.     

Differentiation of Trichoplusia ni MEV and Autographa californica MEV by macrophage migration inhibition tests

The macrophage migration inhibition test (MMI), an in vitro correlate of delayed hypersensitivity, was found to be an effective means of differentiating Trichoplusia ni and Autographa californica multiple embedded nuclear polyhedrosis viruses (NPV). Peritoneal exudate cells from guinea pigs sensitized to virions of T. ni NPV demonstrated significantly different MMI when challenged with T. ni vs A. californica virions. Similarly, when virions of A. californica NPV were employed as the sensitizing antigen, different percentages of MMI were observed in the homologuus versus heterologous challenges.The susceptibility of the fall armyworm, Spodoptera frugiperda, to these two pathogens was very similar as revealed by a comparison of LD₅₀'s, slopes, and fiducial limits of dose-mortality regression lines. In contrast, the cabbage looper, T. ni, was much more susceptible to A. californica NPV.The polyhedral sizes, shapes, and virion occlusion patterns of the two species of NPV were virtually indistinguishable.     

Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution

Baculoviruses are members of a large, well-characterized family of dsDNA viruses that have been identified from insects of the orders Lepidoptera, Hymenoptera, and Diptera. Baculovirus genomes from different virus species generally exhibit a considerable degree of structural diversity. However, some sequenced baculovirus genomes from closely related viruses are structurally very similar and share overall nucleotide sequence identities in excess of 95%. This review focuses on the comparative analysis of partial and complete nucleotide sequences from two groups of closely related baculoviruses with broad host ranges: (a) group I multiple nucleopolyhedroviruses (MNPVs) from a cluster including Autographa californica (Ac)MNPV, Rachiplusia ou (Ro)MNPV, and Plutella xylostella (Plxy)MNPV; and (b) granuloviruses (GVs) from a cluster including Xestia c-nigrum (Xecn)GV and Helicoverpa armigera (Hear)GV. Even though the individual viruses in these clusters share high nucleotide sequence identities, a significant degree of genomic rearrangement (in the form of insertions, deletions, and homologous recombination resulting in allelic replacement) is evident from alignments of their genomes. These observations suggest an important role for recombination in the early evolution and biological characteristics of baculoviruses of these two groups.     

Serological relationships of five nuclear polyhedrosis viruses from lepidopterous species

The serological relationships of five nuclear polyhedrosis viruses (NPV) were investigated using the immunodiffusion technique with intragel absorption. Reciprocal tests demonstrated that virion fractions from Autographa californica multiple embedded virus (MEV), Heliothis armigera MEV, and H. zea single embedded virus (SEV) are not related to each other or to virions from Trichoplusia ni SEV and Pseudoplusia includens SEV. Virion fractions of T. ni and P. includens NPV were shown to be closely related, sharing several antigens. Matrix fractions possessed a common group antigen and one or two antigens specific for the individual NPV with the exception that T. ni and P. includens NPV shared one of these antigens. The specific antigens of the matrix fraction were also shared with the homologous virion fraction.     

Polyacrylamide gel electrophoresis of baculovirus proteins: A simplified and sensitive modification for differentiating between isolates

A rapid and sensitive modification of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of baculovirus structural proteins has been developed. Polyhedral inclusion bodies (PIBs) were pretreated with 1% SDS and 0.5% 2-mercaptoethanol (2-ME) for 30 min at pH 7.2, washed to remove soluble material, dissociated with Laemmli's sample buffer, and analyzed by SDS-PAGE. When the gels were stained with silver nitrate, as little as 48 μg of protein, comprising both polyhedrin and virion proteins, could be resolved on the same gel. Pretreatment with SDS and 2-ME eliminated the need to further purify PIBs by sucrose gradient centrifugation, since gel profiles of PIB proteins before and after such centrifugation were indistinguishable. The method was used to distinguish between the nuclear polyhedrosis viruses (NPVs) of the following species: Mamestra brassicae, Wiseana cervinata, Autographa californica, Mythimna convecta, Persectania dyscrita, Spodoptera exigua, S. frugiperda, Anthela varia, Pterolocera amplicornis, and Heliothis punctiger. Cross-transmission of A. californica NPV to H. punctiger and M. convecta and of M. convecta NPV to P. dyscrita was confirmed by analysis of progeny virus proteins.     

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.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

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.     

Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction.

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. The amino acid sequences of the polyhedrin gene were compared in twenty-six NPVs. A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate PCR primers was designed to produce fragments of about 430 bp. The NPVs detected by this technique were Autographa californica NPV, Bombyx mori NPV, Hyphantria cunea NPV, Spodoptera exigua NPV, S. litura NPV, and Lymantria dispar NPV. This technique would be useful in monitoring the distribution of NPVs and release of the wild type and recombinant NPVs.     

N-terminal polyhedrin sequences and occludedBaculovirus evolution

Summary A phylogenetic tree for occluded baculoviruses was constructed based on the N-terminal amino acid sequence of occlusion body proteins from six baculoviruses including three lepidopteran nuclear polyhedrosis viruses (NPVs), [two unicapsid (Bombyx mori andOrgyia pseudotsugata) and one multicapsid (Orgyia pseudotsugata)]; one granulosis virus (Pieris brassicae); and NPVs from a hymenopteran (Neodiprion sertifer) and a dipteran (Tipula paludosa). Amino acid sequence data for theB. mori NPV were from a report by Sere-bryani et al. (1977) and that for theO. pseudotsugata NPVs were reported previously by us (Rohrmann et al. 1979). The other N-terminal amino acid sequences are presented in this paper. The phylogenetic relationships determined based on the molecular evolution of polyhedrin were also investigated by antigenic comparisons of the proteins using a solid phase radioimmune assay. The results indicate that the lepidopteran NPVs are the most closely related of the above group of viruses and are related to these viruses in the following order:N. sertifer NPV,P. brassicae granulosis virus, andT. paludosa NPV. These data, in conjunction withBaculovirus distribution and evidence concerning insect phylogeny, suggest that theBaculovirus have an ancient association with insects and may have evolved along with them.     

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.     

Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Polyhedral protein preparations from five nuclear polyhedrosis viruses isolated from four closely related host insects of the noctuid subfamily Plusiinae were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), high voltage paper electrophoresis, and amino acid analysis. The viruses were Autographa california multiple-embedded virion type (MEV), Pseudoplusia includens singly embedded virion type (SEV), Rachiplusia ou MEV, Trichoplusia ni MEV, and T. ni SEV. Each was produced in its own host; A. californica MEV was also produced in T. ni larvae to determine possible host influence on polyhedral protein chemistry. Each test revealed minor, reproducible differences among most isolates. In SDS-PAGE, the major protein component ranged from 26,700 to 28,300 MW among the isolates. Differences were confined to minor protein bands or to band intensity. Peptide maps showed differences among most isolates in numbers of acidic and basic peptide spots, but all had an identical number of neutral spots. Migration patterns also differed among most isolates. The amino acid compositions of the six polyhedral inclusions were very similar, with aspartic and glutamic acids being the predominant residues. The greatest differences were found between the MEV and SEV groups, with lesser differences within each group. In all analyses, A. californica MEV produced in A. californica was indistinguishable from virus produced in T. ni.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

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.     

Restriction Map of Rachiplusia ou and Rachiplusia ou-Autographa californica Baculovirus Recombinants

The restriction sites of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. Of the 60 DNA restriction sites of RoMNPV, 35 mapped in similar positions as compared to the restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA. Two plaque-purified viruses, obtained from randomly picked plaques of a wild-type isolate of RoMNPV, were recombinants of RoMNPV and AcMNPV. The recombinants were shown to have RoMNPV and AcMNPV restriction fragments as well as structural polypeptides from each parental virus. Both recombinant viruses had a major RoMNPV capsid protein but were occluded in the AcMNPV polyhedrin protein.