Virion Occlusion Research Articles

Post-mortem incubation influences occlusion body production in nucleopolyhedrovirus-infected larvae of Spodoptera frugiperda

The inefficient production of nucleopolyhedrovirus occlusion bodies (OBs) can limit the commercialization of virus-based insecticides. The production of OBs in SfMNPV-infected Spodoptera frugiperda fourth instars was compared among groups of larvae that were frozen (−20 °C) immediately following death, or subjected to a 9-day period of post-mortem incubation at 5 °C or 15 °C. Incubation at 15 °C resulted in a ∼40% increase in total OB production and OBs per mg larval weight, compared to the −20 °C and 5 °C treatments. OBs from the 5 °C treatment were ∼10% smaller in cross sectional area and small OBs (<1 µm2) were more abundant than in other treatments, possibly due to reduced post-mortem OB maturation in this treatment. SfMNPV genomic DNA in OB samples was 3.6-fold higher in the −20 °C treatment than the 5 °C treatment and 1.7-fold higher than the 15 °C treatment, possibly due to differences in the exposure of viral genomes to degradative enzymes. However, these differences did not affect the concentration-mortality relationship or speed of kill of OBs from the different treatments. The abundance of aerobic microbes increased from ∼3 × 107 CFU/ml in the −20 °C treatment, to approximately 2 × 108 and 3 × 108 CFU/ml in the 5 °C and 15 °C incubation treatments, respectively, similar to levels seen in other nucleopolyhedroviruses produced in insects. We conclude that post-mortem incubation at 15 °C likely involves continuing processes of virion occlusion and OB maturation that increase overall OB production without loss of insecticidal activity, although the value of this step in commercial virus insecticide production will depend on the cost of the incubation step and the value of the additional OBs produced.

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses.

BackgroundPseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated.ResultsThe complete genome of PsinSNPV was sequenced (Roche 454 GS FLX – Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins.ConclusionsPsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1323-9) contains supplementary material, which is available to authorized users.

Cell-dependent production of polyhedra and virion occlusion of Autographa californica multiple nucleopolyhedrovirus fp25k mutants in vitro and in vivo

Members of the family Baculoviridae are insect-specific dsDNA viruses that have been used for biological control of insect pests in agriculture and forestry, as well as in research and pharmaceutical protein expression in insect cells and larvae. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the family Baculoviridae. During infection of AcMNPV in permissive cells, fp25k mutants are positively selected, leading to the formation of the few polyhedra (FP) phenotype with reduced yield of polyhedra and reduced virion occlusion efficiency, which leads to decreased oral infectivity for insects. Here we report that polyhedra of AcMNPV fp25k mutants produced from different insect cell lines and insects have differences in larval per os infectivity, and that these variations are due to different virion occlusion efficiencies in these cell lines and insects. Polyhedra of AcMNPV fp25k mutants produced from Sf cells (Sf21 and Sf9, derived from Spodoptera frugiperda) and S. frugiperda larvae had poorer virion occlusion efficiency than those from Hi5 cells (derived from Trichoplusia ni) and T. ni larvae, based on immunoblots, DNA isolation and larval oral infection analysis. AcMNPV fp25k mutants formed clusters of FP and many polyhedra (MP) in the fat body cells of both T. ni and S. frugiperda larvae. Transmission electron microscopy revealed that the nature of virion occlusion of AcMNPV fp25k mutants was dependent on the different cells of the T. ni fat body tissue. Taken together, these results indicate that the FP phenotype and virion occlusion efficiency of fp25k mutants are influenced by the host insect cells.

Description of a novel single mutation in the AcMNPV polyhedrin gene that results in abnormally large cubic polyhedra

We describe a point mutation in the AcMNPV polyhedrin gene that produces abnormally large cubic polyhedra in packaging cell lines. A polyhedrin mutant baculovirus in which the single change E44G was introduced confirmed that this mutation and no other alterations in the AcMNPV genome was responsible for the abnormal phenotype. Although baculoviral VP39 protein was detected inside mutant polyhedra, electron microscopy demonstrated that only a proportion of the large crystals allow occlusion of virions. When compared with wild-type polyhedra, the mutant inoculum showed reduced oral infectivity for Rachiplusia nu larvae. Hence, the amino acid 44 substitution in the AcMNPV polyhedrin protein alters polyhedrin assembly and affects viral occlusion and infectivity.

Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes

Nucleopolyhedroviruses (NPVs), family Baculoviridae, are insect-specific viruses with the potential to control insect pests in agriculture and forestry. NPVs are occluded in polyhedral occlusion bodies. Polyhedra protect virions from inactivation in the environment as well as assisting virions in horizontal transmission in the insect population. The process of virion occlusion in the polyhedra is undefined and the genes that regulate the virion occlusion process have not been well investigated yet. An Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant (AcDef) that has a 2136 bp DNA deletion, including p26, p10 and p74 genes, has been isolated. No virions were detected in the polyhedra of AcDef. Restoration of all the missing sequences into AcDef led to proper virion occlusion. Individual gene deletion of either p10 or p26 could not abolish virion occlusion in the polyhedra of AcMNPV, but p10 deletion reduced virion occlusion efficiency more than threefold compared with the wild-type AcMNPV. Previous studies by other research groups on deletion of AcMNPV gene p74 suggested that p74 is a per os infectivity factor, and deletion of the p74 gene did not eliminate virion occlusion. Collectively, the three genes (p26, p10 and p74) may act in concert to regulate the virion occlusion process. Therefore, p26, p10 and p74 are all required for proper virion occlusion in the polyhedra of AcMNPV.

Cytopathological process by multiple nucleopolyhedrovirus in the testis of Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

A cytopathological methodology was used to analyze infection by Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), a geographic isolate of the family Baculoviridae, in the caterpillar testes of the B. mori. Japanese B. mori strain caterpillar, fifth instar, were inoculated with BmMNPV and their testes were collected and processed for light and transmission electronic microscopy. Epithelial coating cells and interfollicular septa in testes were susceptible to BmMNPV. The first evidence of infection was detected on the 6th day post-inoculation (p.i.) in the external epithelium, and on the 7th day p.i. in the internal epithelium and interfollicular septa. Cytopathological characteristics consisted of hypertrophied nuclei, the formation of virogenic stroma, and the occlusion of virions in polyhedron protein crystals in several stages of development. At the end of the infectious process, cell lysis and release of polyhedra into the extracellular medium occurred. Histopathology revealed early infection foci in the surrounding regions of tracheal insertions, thus underlining the role of the trachea as an infection-spreading organ in insects. This spreading occurs through penetration of the basal lamina, which facilitates entry of the budded virus into the testis. Additionally, an alignment of a partial sequence of the ORF 14 of the BmMNPV geographic isolate with other NPV certified the virus genera.

PROPERTIES OF A UNIQUE MUTANT OF HELICOVERPA ARMIGERA SINGLE-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS THAT EXHIBITS A PARTIAL MANY POLYHEDRA AND FEW POLYHEDRA PHENOTYPE ON EXTENDED SERIAL PASSAGING IN SUSPENSION CELL CULTURES

Serial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.

Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) replication in Helicoverpa zea serum-free suspension culture was studied in detail and the sequence of virus morphogenesis was determined by transmission electron microscopy. By 16 h post-infection (p.i.), virus replication was observed in the virogenic stroma by the appearance of nucleocapsids. Polyhedron formation was detected by 24 h p.i. and the polyhedron envelope (PE) was completely formed by 72 h p.i. PE morphogenesis of HaSNPV is significantly different compared to the extensively studied Autograph californica (Ac)MNPV. In AcMNPV-infected cells, fibrillar structures are found in both cytoplasm and nuclei, and the fibrillar structures in nuclei are in close association with maturing polyhedra during PE formation. Fibrillar structures that resemble the AcMNPV fibrillar structures were detected only in the cytoplasm of HaSNPV-infected cells and appeared to interact with calyx precursors there, but their role in PE formation is unclear. However, prominent calyx precursor structures of various shapes and sizes were observed in the nuclei of HaSNPV-infected cells as well, and they appeared to interact with polyhedra during PE formation. Both the calyx precursor structure and the cytoplasmic fibrillar structure were detected only after HaSNPV virion occlusion had started, indicating that they might have a role in formation of PE. Similar calyx precursor structures and cytoplasmic fibrillar structures were observed in both serum-supplemented and serum-free suspension cultures, as well as in HaSNPV-infected larval tissues, indicating that the structures observed are not cell culture artefacts.

Characterization of a Single-Nucleocapsid Nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia

A single-nucleocapsid nucleopolyhedrovirus (NPV) isolated from Thysanoplusia orichalcea L. (Lepidoptera:Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1.22 μm (range = 0.803–1.931 μm). The length of the virion with an envelope averaged 0.29 and 0.23 μm without an envelope. ThorNPV was propagated in Pseudoplusia includens (Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV produced in T. orichalcea and P. includens. Polyhedrin amino acid sequence analysis revealed that ThorNPV belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV against third instar P. includens was low, with a LD50 value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions embedded, which might explain the low virulence against P. includens. Differences in virion occlusion rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD50 value to P. includens against Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens, P. includens was the only species infected. The virus infected primarily the fat body, tracheal epithelium, and hypodermis. The genomic size of the ThorNPV is 135 kb.

Site-directed mutagenesis of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin: effect on polyhedron structure

Amino acids Lys34, His36, and Phe37 were substituted by PCR-mediated, site-directed mutagenesis for three Trp's in the AcNPV polyhedrin sequence. Phase contrast microscopy revealed refringent, amorphous polyhedra in the nuclei of infected cells. Electron microscopy confirmed a great variation in form and size of the mutated polyhedra. Although crystallization of the mutated polyhedrin occurred, it was irregular within each polyhedron. Virion occlusion was also severely affected. Virions were partially occluded, or only one virion was occluded per polyhedron. Results suggest that the substitution of these three amino acids affected the morphology of polyhedra, the uniformity of crystallization within each polyhedron, and the virion occlusion.

Specificity of polyhedrin in the generation of baculovirus occlusion bodies.

The role of polyhedrin in the occlusion of virions was studied by substituting two heterologous polyhedrin-coding sequences, one from a multiple-nucleocapsid (M) nucleopolyhedrovirus (NPV) of Spodoptera exigua (Se) and one from a single-nucleocapsid (S) NPV of Buzura suppressaria (BusuNPV), into the genome of Autographa californica (Ac) MNPV. Both heterologous polyhedrin genes were highly expressed and polyhedra were produced in the nuclei of cells infected with the respective recombinant AcMNPVs. Polyhedra produced by the recombinant with BusuNPV polyhedrin showed normal occlusion of multiple-nucleocapsid virions and were equally as infectious to S. exigua larvae as were wild-type AcMNPV polyhedra. This indicates that virion occlusion is not specific with respect to whether the virions or polyhedrin are from an SNPV or MNPV. Polyhedra produced by the recombinant containing the SeMNPV polyhedrin had an altered morphology, being pyramidal rather than polyhedral in shape, and many fewer virions were occluded. These occlusion bodies were less infectious to S. exigua larvae than were those of wild-type AcMNPV. These results indicate that virion occlusion is a finely controlled process that is to some extent specific to the polyhedrin involved and may also require other viral or host factors for optimal morphogenesis.

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.

A baculovirus anti-apoptosis gene homolog of the Trichoplusia ni granulovirus.

An inhibitor of apoptosis (iap) gene homolog (Tn-iap) of the Trichoplusia ni granulovirus (TnGV) was cloned, sequenced and mapped on the genome of TnGV. Tn-iap encoded a protein (Tn-IAP) of 301 amino acids with a predicted molecular mass of 35 kDa. The Tn-IAP contained the two sequence motifs, BIRs and RING finger, characteristic of IAP proteins, and shared identities of 21-27% and similarities of 28-53% with IAP proteins of Cydia pomonella GV (Cp-IAP), Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (MNPV) (Op-IAP1, 3), Autographa californica MNPV (Ac-IAP1), Bombyx mori NPV (Bm-IAP1), Lymantria dispar MNPV (Ld-IAP3) and Buzura suppressaria single nucleocapsid NPV (Bs-IAP1). However, Tn-IAP shared no significant homology with baculovirus IAP2 proteins. Using an antisense Tn-iap probe, two major transcripts of approximately 800 nt and 1600 nt were detected by Northern blot analysis of RNA extracted from the fat body of T. ni larvae infected with the TnGV. Unlike Cp-IAP and Op-IAP3, however, Tn-IAP did not rescue virion occlusion in SF21 cells infected with a p35-deficient AcMNPV mutant. Tn-IAP's synthesis in vivo but failure to rescue p35-deficient AcMNPV in SF21 cells suggests it is a functional IAP that is only effective in certain cell types.

Identification of a NovelLymantria disparNucleopolyhedrovirus Mutant That Exhibits Abnormal Polyhedron Formation and Virion Occlusion

In previous studies on the formation ofLymantria disparnuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in thepolyhedrinor25K FPgene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in thepolyhedringene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include thepolyhedrinor25K FPgenes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.

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 micron) 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.

Polyhedron-like Inclusion Body Formation by a Mutant Nucleopolyhedrovirus Expressing the Granulin Gene from a Granulovirus

The polyhedrin gene inBombyx morinucleopolyhedrovirus (BmNPV) was replaced with the granulin gene ofTrichoplusia nigranulovirus (TnGV). The substitution was verified by Southern hybridization, and expression of granulin by the mutant virus, BmGran, was demonstrated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by amino acid sequencing of the predominant protein of BmGran inclusion bodies (IBs). Light and electron microscopy examination of BmGran-infectedB. moriand BmN cells revealed large, cuboidal, polyhedron-like IBs in the nucleus and cytoplasm, but granules were not seen. IBs contained small, parallel, electron-dense streaks, which defined the geometric pattern of crystallization. Geometric patterns of nuclear IBs were frequently disrupted by occlusion of polyhedron envelope fragments, resulting in IB instability and fracturing. Virions were not embedded in most of the polyhedron-like IBs, but accumulated with polyhedron envelope fragments. Some virions were coated with matrix protein and were partially wrapped by polyhedron envelope. These results suggested that (1) the amino acid sequence of granulin is insufficient for determining IB morphology in TnGV-infected cells, and TnGV may have genes, not present in BmNPV, that control granule formation, and (2) interactions among the virion, the IB envelope, and the matrix protein may be important in virion occlusion and IB morphology and stability.

Identification of a baculovirus polyhedron formation mutant with a novel phenotype

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production.

Serial passage of nuclear polyhedrosis viruses (NPVs) through cultured cell lines results in the appearance of mutants with a complex phenotype referred to as the 'few polyhedra' (FP) phenotype. The altered plaque morphology and reduced occlusion production associated with the FP phenotype have been observed in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) bearing mutations in the gene encoding the 25 kDa protein (25K gene). In this study, we sequenced the 25K genes of four spontaneously occurring AcMNPV FP mutants. These mutants, together with an artificially generated FP mutant (AcFP beta gal, in which the gene for beta-galactosidase is fused in frame with the 25K ORF), were examined at the ultrastructural level to see if they exhibited the reduced virion occlusion and intranuclear envelopment which is associated with the FP phenotype. Observations on Spodoptera frugiperda Sf9 cells infected with the FP mutants revealed that all five mutants were impaired in virion occlusion and intranuclear nucleocapsid envelopment. The 25K mutants were also found to release two- to fivefold more infectious virus (p.f.u.) into the media of infected Sf9 cells. Marker rescue of AcFP beta gal restored wild-type virion occlusion, intranuclear envelopment and levels of infectious virus production.

Rapid Formation of Few Polyhedra Mutants of Lymantria dispar Multinucleocapsid Nuclear Polyhedrosis Virus during Serial Passage in Cell Culture

Four genotypic variants of Lymantria dispar multi-nucleocapsid nuclear polyhedrosis virus (LdMNPV) were used to investigate the generation of few polyhedra (FP) mutants during viral propagation in the L. dispar 652Y cell line. Titers of budded virus, the percentage of infected cells producing polyhedra, the amount of polyhedra produced per cell, the proportion of virus exhibiting an FP phenotype, virion occlusion characteristics, and changes in genomic DNA digestion profiles were investigated through five serial passages. LdMNPV FP mutants arose and became predominate very quickly during serial passage. Greater than 92% of the virus present after the second passage exhibited an FP phenotype in three of the four LdMNPV isolates studied. The amount of budded virus produced by the isolates was from approximately 150- to 250-fold greater at the third passage compared to the amount produced at the first passage. The percentage of infected cells producing polyhedra decreased by an average of 35.6% during serial passage. The number of polyhedra produced per cell decreased between 2.3- and 5.6 fold from the first to the third passage. Restriction endonuclease digestion profiles of the genomic DNA of the FP mutants were analyzed to determine whether genotypic changes occurred that could be correlated with the appearance of the FP phenotype. The genomic DNA digestion profiles of FP mutants in three of the four isolates matched the profiles of the original wild-type isolates. A DNA insertion of 0.9 kb was found in the fourth isolate; however, it was shown not to be the basis for the FP phenotype. These results indicate that FP mutants arise rapidly during serial propagation of LdMNPV in cell culture. In contrast to other baculoviruses, the LdMNPV FP mutants studied in this investigation appear 60 lack DNA insertions of readily detectable length that could be correlated with the appearance of the FP phenotype.