Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture

Linda H. L. Lua1, Marcia R. S. Pedrini1, Steven Reid1, Ashley Robertson2 and David E. Tribe2

Department of Chemical Engineering, The University of Queensland, Queensland 4072, Australia1
Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia2

Author for correspondence: Linda Lua. Fax +617 3365 4199. e-mail lindal{at}cheque.uq.edu.au


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (<10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Serial passaging of baculoviruses in cell culture leads to extensive genomic changes (Miller, 1986 ). The most common and rapidly accumulating mutant detected is a change from the wild-type many polyhedra (MP) per cell phenotype to the few polyhedra (FP) per cell phenotype. The complex FP phenotype is generally described as a decrease in the number of polyhedra produced per cell, few or no virions occluded within polyhedra, altered intranuclear nucleocapsid envelopment and increase in number of infectious budded virus (BV) (Harrison & Summers, 1995b ). FP mutants have been reported in several baculoviruses, including Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) (Hink & Vail, 1973 ), Trichoplusia ni MNPV (TnMNPV) (Potter et al., 1976 ), Galleria mellonella MNPV (GmMNPV) (Fraser et al., 1983 ), Lymantria dispar MNPV (LdMNPV) (Slavicek et al., 1992 ), Orgyia pseudotsugata MNPV (OpMNPV) (Russell & Rohrmann, 1993 ) and Helicoverpa armigera single-capsid NPV (HaSNPV) (Chakraborty & Reid, 1999 ).

Frequent mutations have been identified within a specific region (map units 35·0–37·0) in the FP mutants of AcMNPV (Beames & Summers, 1988 , 1989 ; Fraser et al., 1983 ) and GmMNPV (Fraser et al., 1985 , 1983 ). This region contains the 25K FP gene, which encodes a 25 kDa protein that is essential for virion occlusion and polyhedron formation (Beames & Summers, 1988 , 1989 ; Wang et al., 1989 ). Most reports on FP mutations were correlated to large insertions of host DNA or deletions of the viral genome (0·1–2·8 kb), which were detectable by restriction endonuclease (REN) digestion analysis (Beames & Summers, 1988 , 1989 ; Fraser et al., 1985 , 1983 ; Wang et al., 1989 ). The FP mutations of LdMNPV were exceptions (Bischoff & Slavicek, 1997 ; Slavicek et al., 1995 ). These LdMNPV FP mutants exhibit FP characteristic traits; however, REN digestion analysis did not correlate the FP phenotypes observed to any DNA insertions or deletions of detectable lengths. Bischoff & Slavicek (1997) later reported 1 bp insertions or small deletions (8 or 24 bp) in the 25K FP gene of LdMNPV FP mutants, suggesting that the mechanism of FP mutation in LdMNPV could be different from that of AcMNPV.

In recent years, studies have been done to determine the role of the 25 kDa protein in the infection cycle of baculoviruses. However, the analysis of the function of the 25 kDa protein is not conclusive (Beniya et al., 1998 ; Braunagel et al., 1999 ; Harrison & Summers, 1995a , b ). Studies showed that the 25K FP protein is a structural protein in the nucleocapsids of both BV and occlusion derived virus (ODV) but a large fraction of the 25K FP proteins remains cytoplasmic throughout infection (Braunagel et al., 1999 ; Harrison & Summers, 1995a ). The relationship between the location of the 25K FP gene product and the phenotype caused by the gene mutation is still unclear. Considering the varied effects following mutation of this gene, it is possible that the gene has more than one function during the invasion and infection process. Studies also suggest that the 25K FP protein could be required for efficient protein accumulation and trafficking by regulating transcription, mRNA stability, translation or altered protein stability of one or many viral proteins, which directly or indirectly affect occlusion morphogenesis (Beniya et al., 1998 ; Braunagel et al., 1999 ).

Reduced occlusion production and decreased virulence as a result of FP mutations would greatly hinder the commercialization of baculoviruses produced by in vitro cell culture, for use as biopesticides. As large volumes of virus inocula are needed for large-scale production of baculoviruses (Rhodes, 1996 ), it may be impossible to generate enough virus inocula at low passage numbers for such production, as emergence of FP mutants appears to be inevitable during the scale-up of virus inocula used for cell culture systems. Understanding the nature of FP mutations and how they are selected may help in either developing a process for isolating virus isolates with a stable MP phenotype or to minimize the selective advantage of FP mutants. Research on FP mutation not only deserves further attention from the commercial perspective but also for its intrinsic interest.

Chakraborty & Reid (1999) reported the effect of serial passaging on HaSNPV in cell culture; however, no insights were given on the phenotype or genotype of HaSNPV FP mutants. The current paper reports detailed phenotypic and genotypic studies of HaSNPV FP mutants. HaSNPV was serially passaged in Helicoverpa zea serum-free suspension cultures and the phenotypic changes that occurred during the transition of an MP dominated population to a FP one was investigated using transmission electron microscopy (TEM). The ultrastructural differences between the MP and FP infected cells during progeny virus production and assembly were documented. REN digestion profiles of genomic viral DNA during serial passaging were determined, the wild-type HaSNPV 25K FP gene was identified and sequenced, and a FP mutant that carries a mutation in the 25K FP gene was isolated.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and virus.
The Helicoverpa zea cell line was obtained from CSIRO, Division of Entomology (Canberra, Australia). It was isolated from pupal ovarian tissue, strain BCIRL-HZ-AM1 (McIntosh & Ignoffo, 1981 ). The uncloned cells were gradually adapted to serum-free SF900II (Life Technologies) suspension culture (Chakraborty, 1997 ). Cultures were typically grown in 250 ml Erlenmeyer flasks on an orbital shaker operated at 120 r.p.m., inside a refrigerated incubator at 28 °C. HaSNPV (uncloned) passage 1 stock was established in cells grown in SF900II supplemented with 10% foetal bovine serum (CSL, Australia), with haemolymph collected from infected larvae which were obtained from the Department of Primary Industries (Brisbane, Queensland, Australia). The details are documented in an earlier paper (Lua & Reid, 2000 ).

{blacksquare} Serial passaging of HaSNPV in vitro.
For all passages, duplicate 100 ml working volumes of Helicoverpa zea cell cultures at 5x105 cells/ml were infected at an m.o.i. of 0·5 p.f.u. per cell. All cultures were seeded at 3x105 cells/ml (50 ml volumes), and allowed to grow to 1x106 cells/ml, before being diluted back to 5x105 cells/ml with 50 ml fresh medium (100 ml final volume). To obtain infectious BV for subsequent passage, 50 ml of the cell suspension was harvested at 70% viability, typically 4 or 5 days post-infection (p.i.), leaving the other 50 ml cell suspension for polyhedra production. All virus stocks were stored in 1·8 ml or 4 ml aliquots at -70 °C. The virus titre of each passage was determined using a plaque assay (Lua & Reid, 2000 ), before use as inoculum for the next passage. Cell densities and cell viabilities of all cultures were determined daily in triplicate using the 0·1% trypan blue exclusion cell count method (Nielsen et al., 1991 ). Final polyhedra yields were determined at 10 days p.i. Cells were first lysed with 0·5% SDS for 1 h at 28 °C before triplicate counts of the polyhedra were determined using a haemocytometer counting chamber.

{blacksquare} Plaque purification of HaSNPV FP mutant.
A second HaSNPV population (uncloned) was established in cell culture with collected haemolymph under similar conditions to those described above. The virus was serially passaged out to passage 6. BV at passage 6 was diluted out onto culture plates as in a plaque assay and an FP plaque was picked on the basis of its phenotypic appearance under light microscopy (Hink & Vail, 1973 ). The FP mutant (designated as FP8AS) was plaque purified three times before being scaled up to obtain BV DNA for PCR amplification and sequencing. The FP phenotype of the plaque purified mutant was confirmed using TEM.

{blacksquare} TEM and percent of FP infected cells.
At each passage, infected cell pellets were harvested at 6 days p.i. for determination of percentage of FP/MP using TEM. Cell suspension (1 ml) was centrifuged at 12000 r.p.m. for 10 min. The cell pellets were resuspended in 3% glutaraldehyde fixative solution and kept at 4 °C. The protocol for TEM processing is documented in Lua & Reid (2000) . The% FP infected cells was determined by assessing m infected cells under TEM and scoring each cell as either a FP infected cell or an MP infected cell. If we assume that each observation is independent, the number of FP infected cells observed, x, will be binomially distributed with the probability p equal to the% FP in the population (Taylor, 1997 ). The confidence interval for p is given by:

{blacksquare} Bioassay.
The bioassay used to estimate the potency of the polyhedra produced against Helicoverpa armigera larvae measured the concentration of polyhedra required to kill a larva. The potency is usually expressed as LC50 (median lethal concentration), which is the concentration of polyhedra required to kill 50% of the population. Infected cells with polyhedra at 10 days p.i. were harvested and kept frozen at -20 °C for bioassay. Polyhedra were extracted and bioassays were performed by Peter Christian (CSIRO Entomology, Canberra, Australia). The potency of the polyhedra to be tested was compared to a standard virus, GemStar® [Helicoverpa zea (Hz)SNPV produced in vivo], by simultaneously testing both.

{blacksquare} REN digestion analysis.
BV DNA from passage 2 to passage 6 was purified for REN digestion analysis. To purify DNA from BV, cells were first removed by low speed centrifugation (1000 g, 10 min). The BV was pelleted by ultracentrifugation of the supernatant at 10000 g for 45 min at 4 °C, using an ultracentrifuge with an SW28 rotor (Centrikon T-2070, Kontron Instruments) and 38·5 ml Ultra-Clear centrifuge tubes (Beckman). Each BV pellet was resuspended with 200 µl cold PBS. DNA was purified from the BV using the QIAamp DNA Blood Mini Kit (Qiagen) according to the protocol provided by the manufacturer. The viral DNA was digested separately with EcoRI, HindIII and BamHI, at 37 °C for 4 h. Each digestion consisted of 25 µl viral DNA, 2 µl enzymes and the appropriate buffer for specific restriction enzyme requirements. The fragments were separated by electrophoresis on a 0·7% agarose gel at 60 W for 2 to 3 h and stained with ethidium bromide. Digital images of the agarose gels were obtained using a Kodak EDAS 120 digital camera with software provided (Kodak Digital Science 1D, version 3.0.0).

{blacksquare} PCR amplification and sequencing of the 25K FP gene.
This work was performed before the entire genome of HaSNPV was published (Chen et al., 2001 ). A set of upstream and downstream degenerated oligonucleotides was first designed from multiple alignment of the 25K FP gene of different baculoviruses and used to identify the 25K FP gene of HaSNPV. They were oligonucleotides 5' KGYAGYGTNGARATFTAYGG 3' (Primer 1) and 5' NATYTTNACNGGNCCRTCRTA 3' (Primer 2). The reaction buffer supplied by the manufacturer of the Taq DNA polymerase (Boehringer Mannheim) was used with dNTPs (Pharmacia) at a final concentration of 200 µM. The amplification cycle consisted of 3 min denaturation at 94 °C, annealing for 1 min at 45 °C and extension at 72 °C for 1 min. The amplification cycle was repeated 30 times. Specific oligonucleotides, 5' CGGTATTCACGATAGAAA 3' (Primer 3) and 5' CGTAGTCTATGTCTAGAT 3' (Primer 4), were designed to confirm the nucleotide sequence of the first identified fragment of the 25K FP gene. Primers 3 and 4 were also used for primer walking sequencing, using the purified genomic viral DNA as template. A 40 µl sequencing reaction consisting of 16 µl BigDye premix, 13 pM primer and 200–300 ng viral genomic DNA was set up: 99 cycles consisting of 30 s at 95 °C for denaturation of template, 20 s at 50 °C for annealing of primer and 4 min at 60 °C for extension of nucleotides were operated. The final products were cleaned up with the Centrispin-20 columns (Geneworks), according to the protocol provided by the manufacturer, before being sequenced. Oligonucleotides 5' GTACGCACACATATACAC 3' (Primer 5) and 5' GCTAGTCAAATGAGTCGC 3' (Primer 6) were used to amplify and sequence the entire 25K FP gene. Oligonucleotides 5' ACGGACTGGATGAGCTTC 3' (Primer 7) and 5' CGGTACTCGGTAAATCTG 3' (Primer 8) were used to amplify and sequence the entire 25K FP gene including upstream and downstream regions of the gene. The annealing temperature used in the PCR amplification cycles for Primer 3 to Primer 8 was 50 °C. The nucleotide sequence of amplified DNA was determined directly. Sequencing was performed using the PRISM (Applied Biosystems) terminator chemistry method according to the manufacturer’s instructions: 30–90 ng DNA and 3·2 pM primer in a 16 µl reaction was prepared for sequencing, whilst the other reagents for the reaction were supplied in the PRISM kit. Reaction conditions (including amplification cycle) and the removal of unincorporated reaction components were performed according to the manufacturer’s recommendations. Sequence analysis were done with SEQUENCHER (Genecodes Corp.). All nucleotide sequences were determined on both strands from at least two independently amplified templates and consensus sequences were obtained from multiple determinations to avoid errors arising from the use of Taq polymerase.

{blacksquare} Cloning PCR products.
Amplicons of the 25K FP gene of passage 6 BV were cloned into pGEM-T Vector (Promega) and transformed into E. coli JM109 competent cells. After transformation, the positive clones were picked and DNA templates for sequencing were isolated using the Qiagen Plasmid Mini Kit.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Serial passaging of HaSNPV in vitro
The effect of serial passaging HaSNPV in Helicoverpa zea suspension cultures was investigated through analysis of the cell specific yield (polyhedra produced per cell), BV production and percentage of FP infected cells at each passage as observed using TEM. A decrease in cell specific yield was noted as the HaSNPV was serially passaged in SF900II medium (Fig. 1). The drop was most pronounced from passage 3 to passage 4. In a single passage, the cell specific yield decreased from 178 polyhedra per cell to 57 polyhedra per cell, a 65–70% reduction in yield (Table 1). By the sixth passage, the cell specific yield was less than 5 polyhedra per cell. In contrast to the drop in polyhedra yield from the third to the fourth passage, the BV titre increased 3-fold. However, the BV titres declined from passage 5 onwards. TEM examination of the infected cells revealed an increase in the percentage of FP infected cells with increasing passage number. A steep increase in the FP mutant population was noted from the third passage to the fourth passage. The steep slope between passage 3 and passage 4 indicates a sharp transition from an MP dominated population to a FP one.



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Fig. 1. Serial passaging of HaSNPV in SF900II. The polyhedra cell specific yield ({bullet}), budded virus titre ({blacktriangleup}) and percent FP infected cells ({blacksquare}) at each passage were determined.

 

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Table 1. Serial passage data for HaSNPV in SF900II medium

 
Bioassays were performed to determine the biological activity of polyhedra from the serially passaged HaSNPV. The biological activity of the polyhedra decreased with passage number (Table 1). Passage 4 polyhedra were similar in potency to passage 3 polyhedra; however, passage 6 polyhedra were 28-fold less potent compared to passage 3 polyhedra.

Nucleocapsid envelopment and virion occlusion
In a wild-type MP infected cell, nucleocapsids are produced in the virogenic stroma, and migrate to the ring zone of the nucleus to acquire their envelopes through a de novo intranuclear membrane synthesis process (Fig. 2A). The enveloped virions then gather in the ring zone, near the inner nuclear membrane, ready for occlusion into the polyhedrin protein matrix. Abnormalities in morphogenesis became apparent at the fourth passage. A majority of the nucleocapsids in the ring zone of the FP infected cells did not acquire envelopes (Fig. 2B). The intranuclear membrane profiles believed to be involved in the nucleocapsid envelopment process were clearly observed in these FP infected cells. However, only infrequently were the nucleocapsids enveloped in the FP infected cell cases, indicating an impaired nucleocapsid envelopment process in FP infected cells.



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Fig. 2. TEM images showing phenotypic characteristics of MP (passage 3) and FP (passage 4) infected cells. (A) Enveloped virions in the ring zone of the nucleus (an MP infected cell). (B) Naked nucleocapsids (arrowheads) accumulating in the ring zone of a FP infected nucleus with presence of de novo intranuclear membrane profiles (arrows). (C) Virion occlusion in MP producer. (D) FP mutant producing polyhedra without any virions occluded. (E) MP producer with many polyhedra in the nucleus and all polyhedra have virions occluded. (F) Few polyhedra were produced by the FP infected cell and the polyhedra formed have no virions occluded. Nu, nucleus; VS, virogenic stroma; r, ring zone; v, virion; nc, nucleocapsid; pp, polyhedrin; p, polyhedron; nm, nuclear membrane; c, cytoplasm.

 
Examination of the occlusion process in MP infected cells suggests that the enveloped virions were progressively occluded by polymerization of polyhedrin protein (Fig. 2C). Naked, unenveloped nucleocapsids did not appear to be occluded by deposition of polyhedrin protein on their surface. Few polyhedra were produced in FP infected cells, and usually these polyhedra do not have virions occluded (Fig. 2D). Under TEM, a reduction in the number of polyhedra per section of a FP infected cell, as compared to an MP infected cell, was clearly detected (Fig. 2E, F).

Obvious signs of infection, such as presence of virogenic stroma, nucleocapsids and intranuclear membrane profiles, were evident in the FP infected cells. Despite the signs of infection, some FP infected cells do not form polyhedra (Fig. 3A). Another phenotypic characteristic of FP infected cells was accumulation of intranuclear membrane profiles in the ring zone of infected nuclei (Fig. 3B). In some FP mutant cases, the intranuclear membranes became elongated, angular and unusual in shape, forming massive swirls in the ring zone of the nuclei. In addition, some FP infected cells also produced polyhedra with morphologically aberrant virions occluded (Fig. 3C) or associated with the edge of an occlusion (Fig. 3D), or polyhedra with very few virions occluded (Fig. 3E).



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Fig. 3. Appearance of various FP infected cells at passage 4 and passage 5. (A) Infected cell with many nucleocapsids and intranuclear membrane profiles (arrow) but no formation of polyhedra. (B) Massive swirls or whorls in the ring zone of nucleus. (C) Polyhedron with aberrant morphology. (D) Non-enveloped and enveloped virions failed to be occluded during the crystallization of polyhedrin. (E) Aberrant polyhedra with few virions occluded. VS, virogenic stroma; nm, nuclear membrane; nc, nucleocapsid; p, polyhedron; v, virion.

 
REN digestion analysis
The genomic REN digestion patterns of passage 2 to passage 6 BV DNA were obtained with three restriction endonucleases to determine if genomic changes had occurred (such as large insertions or deletions) during serial passaging that could be correlated to the appearance of the FP phenotype. The REN digestion patterns of passage 2 to passage 6 virus appeared similar (Fig. 4). No large insertion or deletion was indicated as likely from the REN digestion patterns generated by any of the restriction endonucleases.



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Fig. 4. Genomic DNA digests of HaSNPV with BamHI (A), EcoRI (B) and HindIII (C). The lanes marked M contain DNA size markers (1 kb DNA ladder) and lanes marked P2–P6 contain digests of genomic DNA obtained from budded virus after each serial passage.

 
Genetic analysis of the 25K FP gene of wild-type HaSNPV and a FP mutant
The phenotypic observations on the HaSNPV FP mutants suggest mutations in the 25K FP gene as reported for other baculoviruses. Thus, the 25K FP gene of HaSNPV was identified and sequenced. A small fragment (250 bp) of the 25K FP gene was first amplified and sequenced with a set of degenerate primers that were designed based on the conserved region of the 25K FP gene for five other baculoviruses. The entire 25K FP gene nucleotide sequence was obtained with primer walking sequencing directly on the genomic DNA template. The 25K FP gene opening reading frame of HaSNPV is 654 bp, encoding 217 amino acids (Fig. 5A). Seven nucleotide differences (at nucleotide positions 45, 124, 288, 426, 561, 570 and 576, as starting from the first nucleotide of the gene) were detected between the 25K FP gene nucleotide sequences reported in this paper and the HaSNPV genome recently published by Chen et al. (2001) . No predicted amino acid difference was observed for all nucleotide differences except at nucleotide position 576, which results in a conservative residue change (an aspartate for our isolate whereas it is a glutamate for Chen’s group). The amino acid sequence of the 25K FP gene is conserved among baculoviruses (Fig. 5B). The only region lacking significant conservation is the last 11–35 C-terminal amino acids.



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Fig. 5. Genetic analysis of the HaSNPV 25K FP gene. (A) Nucleotide sequences of the 25K FP gene including the upstream and the downstream regions of the gene. The 25K FP gene is 654 bp (bold). The region that contains a string of seven adenine residues is underlined. (B) Multiple amino acid sequence alignment of 25K FP protein from various nucleopolyhedroviruses. The number of amino acids encoded by each gene is indicated at the end of each sequence. AcMNPV (Ayres et al., 1994 ); BmMNPV (Gomi et al., 1999 ); GmMNPV (Beames & Summers, 1989 ); LdMNPV (Bischoff & Slavicek, 1997 ); OpMNPV (Ahrens et al., 1997 ); SeMNPV (IJkel et al., 1999 ).

 
The nucleotide sequence of the 25K FP gene was determined from the wild-type HaSNPV (in vivo produced ODV), passage 2 BV and passage 6 BV for comparison. Contrary to expectations, no nucleotide differences within the 25K FP gene were detected in any of these samples. Even the upstream and downstream regions of the gene (200–250 bp on each end) were identical for all sources of DNA. Clean chromatographic sequence data without overlapping or hidden peaks were obtained from all sequencing samples. As it was possible that passage 6 BV might be composed of a mixture of FP and MP viruses that appeared to lack detectable DNA insertions or deletions within this gene in the consensus sequences obtained from the amplicons, further sequence work was warranted. The 25K FP gene amplicons of passage 6 BV were cloned: nine individual clones were picked and sequenced to measure the variability of this gene at passage 6. A three times plaque-purified FP mutant (FP8AS) was also isolated. As presented in Fig. 6(A), many point mutations and/or point insertions were observed throughout the entire 25K FP gene. Only two out of the nine clones were found to carry the wild-type 25K FP gene sequence.



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Fig. 6. Mutations in the HaSNPV 25K FP gene. (A) Different mutations identified in the 25K FP gene from the clones of the 25K FP gene PCR products. Point mutations were found randomly throughout the entire gene. (B) Predicted effects of the mutations on the 25K FP protein amino acid sequences expressed by the HaSNPV FP mutants. Point mutations at nucleotide position 102 (clones C8 and C44) would not result in a predicted amino acid change; 1 bp insertions (FP8AS, clones C2 and C48) result in a coding frameshift and truncated proteins. Solid areas indicate homologous regions of the mutant compared to the wild-type protein and the hatched area indicates non-homologous regions.

 
Four clones (C8, C19, C52 and C59) were identified to contain one or two point mutations, which are predicted to result in a single amino acid change. No amino acid change was predicted for clone C44, which contains a single nucleotide change in position 102 (C8 also carries this change along with a change at nucleotide position 218). Clone C48 has an extra adenine residue inserted within a string of six adenine residues between nucleotide position 256 and 262, whereas an extra adenine residue was inserted within a string of seven adenine residues (nucleotide position 418–424) in both FP8AS and clone C2. These insertions found on FP8AS, clones C2 and C48, result in a coding frameshift, leading to the introduction of stop codons (after amino acid 143 for FP8AS and clone C2, after amino acid 112 for clone C48), which would then result in truncated proteins compared to the wild-type 25K FP protein (Fig. 6B). Electron microscopy has also confirmed that the isolate FP8AS exhibited typical phenotypic characteristics of an FP mutant, such as few polyhedra per cell and polyhedra without virions occluded.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
HaSNPV appears to undergo rapid mutation and selection events when serially passaged in cell culture, leading to the formation and selection of FP mutants. The HaSNPV isolate was predominately a population of MP virus at passage 1 in culture but transformed totally to a FP population after six passages. This transition was obvious between passage 3 to passage 4 as a 65–70% reduction in cell specific yield and an 80% increase in FP mutants was observed within one passage (Fig. 1). Upon more careful examination, these FP mutants exhibit characteristic traits such as low polyhedra production (on average less than 10 polyhedra per ultrathin section), reduced or no virions occluded, an impaired intranuclear nucleocapsid envelopment process and reduced potency (Fig. 2). The phenotypic effects of FP mutations observed with HaSNPV are similar to those previously reported for other baculoviruses such as AcMNPV (Harrison & Summers, 1995b ), TnMNPV (Potter et al., 1976 ), GmMNPV (Fraser et al., 1983 ), Bombyx mori MNPV (BmMNPV) (Katsuma et al., 1999 ), and LdMNPV (Slavicek et al., 1992 ).

The number of polyhedra produced by a HaSNPV FP infected cell was significantly lower (<10 polyhedra per section) than a HaSNPV MP infected cell ( 50 polyhedra per section) (see Table 1 and Fig. 2). Harrison et al. (1996) demonstrated that the rate of polyhedrin mRNA expression is significantly reduced in cells infected with AcMNPV FP mutants compared to that of an MP infection. In FP infected cells, polyhedrin localizes less efficiently to the nucleus during the early occlusion phase of infection (24 h post-infection), yet polyhedrin mRNA stability is similar in MP and FP mutant infected cells. The reduction in polyhedrin synthesis and nuclear localization could account for the reduced number of polyhedra observed with HaSNPV FP mutant infections and other baculoviruses by resulting in a significantly decreased concentration of polyhedrin available in the nucleus for occlusion assembly. Clearly, this requires further investigation of the effect of the 25K FP gene mutation on the expression and localization of polyhedrin in the HaSNPV infected cells.

An accumulation of a large number of naked nucleocapsids in the nuclei of FP infected cells, presumably a result of an impaired nucleocapsid envelopment, was a striking observation in this study. Although nucleocapsid assembly is apparently normal, there are few enveloped virions (the ODV) present in FP mutant infected cells. The appearance of intranuclear accumulations of short, open-ended, membrane profiles in the FP mutant infected cells also suggested that there is a specific failure in the envelopment process of nucleocapsids, not in the production of envelopes themselves. Altered intranuclear nucleocapsid envelopment due to FP mutation in AcMNPV was reported as early as 1974 (Ramoska & Hink, 1974 ). Naked nucleocapsids did not appear to be occluded. It is well documented that only enveloped virions are associated with polyhedrin protein, leading to occlusion of the virions (Harrap, 1972 ). The virion envelope (or proteins embedded in it) may be necessary for an interaction of the virion with polyhedrin protein during the occlusion process (Wood, 1980 ). Thus, an impaired intranuclear nucleocapsid envelopment process in HaSNPV FP mutant infected cells may directly or indirectly result in the production of FP polyhedra that have no virions occluded.

The rate of FP mutant proliferation relative to parental type in different cell–virus systems is variable. In this work, more than 80% of cells were infected by FP mutants at passage 4 during serial passaging of HaSNPV, and 100% FP mutant infected cells were obtained by passage 6. With TnMNPV, the virus population was 90% FP only after 10 passages (Potter et al., 1976 ) but mutation/selection was much faster for LdMNPV, which shows a 96% FP population after passage 2 (Slavicek et al., 1995 ). OpSNPV FP mutation is affected by host cells (Sohi et al., 1984 ), but formation of FP variants of GmMNPV is not a host-dependent phenomenon and their detection can be influenced by the overlay formulation used during plaque assays (Fraser & Hink, 1982 ). This suggests that differences in media used to propagate cells and prepare overlays may contribute to inconsistencies in the quantitative detection of FP plaques. FP mutants are often distinguished from MP virus on the basis of a less refractive plaque morphology, which can be subjective. Therefore, in this study, TEM was used to score MP and FP mutant infected cells at each passage instead of conventional plaque assays.

A trait commonly observed in most baculovirus FP mutations is an increase in the production of BV. Higher BV titres were reported in FP mutants of AcMNPV (Harrison & Summers, 1995b ; Wood, 1980 ), TnMNPV (Potter et al., 1976 , 1978 ) and LdMNPV (Slavicek et al., 1995 ). The BV titres of LdMNPV FP mutants increase significantly, as high as 150- to 250-fold (Slavicek et al., 1995 ). However, an increase of BV production due to FP mutation was not observed for HaSNPV. A significant 3-fold increase in the BV titre was only detected at the fourth passage of the HaSNPV isolate, but decreased in the subsequent passages. Chakraborty & Reid (1999) reported a steady increase in BV titres from passage 2 to passage 5 during serial passaging of HaSNPV in SF900II medium supplemented with 10% serum but titres decreased in the subsequent passages. Interestingly, declining BV titres during serial passaging of HzSNPV (Yamada et al., 1982 ) and LdMNPV (Lynn, 1994 ) were also reported. A decrease in BV production during serial passaging in cell culture could either be a trait specific to the Helicoverpa sp. or may be dependent on the cell–virus system. As HaSNPV FP mutations do not cause an increase in BV production, one possible reason for the FP mutants to out-complete MP virus could be their ability to bind and infect cells at a higher efficiency than MP virus.

The phenotypic observations on the HaSNPV FP mutants suggest genomic changes occurring in the 25K FP gene, which could be large insertions or deletions within the gene. However, the genomic digestion profiles of all passages generated separately with three restriction endonucleases were similar, indicating that no large insertions or deletions of the viral genome occurred during serial passaging. The consensus sequence data obtained from the 25K FP gene amplicon of both passage 2 and passage 6 material are identical. This result was unexpected since the HaSNPV FP mutants exhibit well-documented FP phenotypes that are correlated to mutations in the 25K FP gene of other baculoviruses. Speculations on the possibility that passage 6 comprised a mixture of FP mutants that carry very small insertions or deletions as a result of random mutations, thus resulting in a consensus sequence of the wild-type 25K FP gene, was confirmed when 25K FP gene sequences obtained from individual clones of passage 6 amplicons revealed a mixture of point mutations and/or point insertions within the gene. Two out of nine clones (C2 and C48) carried 1 bp insertions within a region of adenine repetitive sequences. Five other clones carried point mutations on the gene. Further investigation with a plaque-purified FP mutant (FP8AS) also revealed a 1 bp insertion in the same region of repetitive sequences as clone C2 within the 25K FP gene. Frameshift mutations as a result of point insertions in FP8AS, clones C2 and C48 would lead to truncated 25K FP proteins. The 25K FP gene mutations observed in HaSNPV are not specific to a particular region but rather they are randomly found throughout the gene. Hence, not all baculovirus FP mutants have similar genetic mutations within the 25K FP gene.

The FP mutation of HaSNPV is likely to arise through a different mechanism from AcMNPV and GmMNPV (Beames & Summers, 1988 ; Fraser et al., 1983 ) but probably similar to that for LdMNPV (Bischoff & Slavicek, 1997 ; Slavicek et al., 1995 ). HaSNPV FP mutants are not generated through large DNA insertions or deletions that are of sufficient size to allow detection by genomic REN digestion analysis. Five nucleotide sites consisting of 5' TTAA 3', which are frequently associated with transposon insertions within the AcMNPV and GmMNPV 25K FP genes (Beames & Summers, 1988 ; Fraser et al., 1985 ; Wang et al., 1989 ), were found in the HaSNPV 25K FP gene. However, 14 such sites were identified in the AcMNPV 25K FP gene (Wang et al., 1989 ), which could lead to a higher rate of transposon insertions for AcMNPV. One bp insertions in repetitive sequences and point mutations observed for HaSNPV are more similar to the 25K FP gene mutations reported for LdMNPV (Bischoff & Slavicek, 1997 ). As discussed by Bischoff & Slavicek (1997) , 1 bp insertions within repetitive sequences (observed here with isolate FP8AS, clones C2 and C48) are most likely the result of DNA polymerase slippage on the newly synthesized strand.

The work has further confirmed the importance of the 25K FP gene during the production and assembly of progeny virus. From both a fundamental and applied science perspective, the effects of mutation in this gene deserve further study. Characterization of HaSNPV MP and FP virus is currently under way in our laboratory, in an effort to understand the selective advantage of HaSNPV FP mutants since they do not appear to gain their advantage simply through the production of higher BV titres. In particular, the cell–virus binding and uptake characteristics of MP and FP virus are under investigation.


   Acknowledgments
 
We are grateful for the bioassays carried out by Dr Peter Christian (CSIRO) and the technical advice provided by the Centre for Microscopy and Microanalysis (UQ). We also thank Dr Lars Nielsen for his help with statistical analysis. This research was supported by the Australian Research Council and the Grains Research and Development Corporation. Scholarships were provided by the University of Queensland and the Brazilian Ministry of Education and Culture.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AF395841 and AF395871.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Ahrens, C. H., Russell, R. L., Funk, C. J., Evans, J. T., Harwood, S. H. & Rohrmann, G. F. (1997). The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology 229, 381-399.[Medline]

Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586.[Medline]

Beames, B. & Summers, M. D. (1988). Comparisons of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra baculovirus mutants. Virology 162, 206-220.[Medline]

Beames, B. & Summers, M. D. (1989). Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants. Virology 168, 344-353.[Medline]

Beniya, H., Braunagel, S. C. & Summers, M. D. (1998). Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes. Virology 240, 64-75.[Medline]

Bischoff, D. S. & Slavicek, J. M. (1997). Phenotypic and genotypic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in 25K FP gene may be caused by DNA replication errors. Journal of Virology 71, 1097-1106.[Abstract]

Braunagel, S. C., Burks, J. K., Rosas-Acosta, G., Harrison, R. L., Ma, H. & Summers, M. D. (1999). Mutations within the Autographa californica nucleopolyhedrovirus FP25K gene decrease the accumulation of ODV-E66 and alter its intranuclear transport. Journal of Virology 73, 8559-8570.[Abstract/Free Full Text]

Chakraborty, S. (1997). In vitro production of wild-type Helicoverpa baculovirus. PhD thesis, University of Queensland, Australia.

Chakraborty, S. & Reid, S. (1999). Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures. Journal of Invertebrate Pathology 73, 303-308.[Medline]

Chen, X., IJkel, F. W. J., Tarchini, R., Sun, X., Sandbrink, H., Wang, H., Peters, S., Zuidema, D., Lankhorst, R. K., Vlak, J. M. & Hu, Z. (2001). The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. Journal of General Virology 82, 241-257.[Abstract/Free Full Text]

Fraser, M. J. & Hink, W. F. (1982). Comparative sensitivity of several plaque assay techniques employing TN-368 and IPLB-SF 21AE insect cell lines for plaque variants of Galleria mellonella nuclear polyhedrosis virus. Journal of Invertebrate Pathology 40, 89-97.

Fraser, M. J., Smith, G. E. & Summers, M. D. (1983). Acquisition of host cell DNA sequences by baculoviruses: relationship between host DNA insertions and FP mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses. Journal of Virology 67, 287-300.

Fraser, M. J., Brusca, J. S., Smith, G. E. & Summers, M. D. (1985). Transposon-mediated mutagenesis of a baculovirus. Virology 145, 356-361.[Medline]

Gomi, S., Majima, K. & Maeda, S. (1999). Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. Journal of General Virology 80, 1323-1337.[Abstract]

Harrap, K. A. (1972). The structure of nuclear polyhedrosis viruses. III. Virus assembly. Virology 50, 133-139.[Medline]

Harrison, R. L. & Summers, M. D. (1995a). Biosynthesis and localization of the Autographa californica nuclear polyhedrosis virus 25K gene product. Virology 208, 279-288.[Medline]

Harrison, R. L. & Summers, M. D. (1995b). 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. Journal of General Virology 76, 1451-1459.[Abstract]

Harrison, R. L., Jarvis, D. L. & Summers, M. D. (1996). The role of the AcMNPV 25K gene, ‘FP25,’ in baculovirus polh and p10 expression. Virology 226, 34-46.[Medline]

Hink, W. F. & Vail, P. V. (1973). A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line. Journal of Invertebrate Pathology 22, 168-174.

IJkel, W. F. J., Van Strien, E. A., Heldens, J. G. M., Broer, R., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1999). Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. Journal of General Virology 80, 3289-3304.[Abstract/Free Full Text]

Katsuma, S., Noguchi, Y., Zhou, C. L., Kobayashi, M. & Maeda, S. (1999). Characterization of the 25K FP gene of the baculovirus Bombyx mori nucleopolyhedrovirus: implications for post-mortem host degradation. Journal General Virology 80, 783-91.[Abstract]

Lua, L. H. L. & Reid, S. R. (2000). Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture. Journal of General Virology 81, 2531-2543.[Abstract/Free Full Text]

Lynn, D. E. (1994). Enhanced infectivity of occluded virions of the gypsy moth nuclear polyhedrosis virus for cell culture. Journal of Invertebrate Pathology 63, 268-274.

McIntosh, A. H. & Ignoffo, C. M. (1981). Replication and infectivity of the single-embedded nuclear polyhedrosis virus, baculovirus Heliothis, in homologous cell line. Journal of Invertebrate Pathology 37, 258-264.

Miller, L. K. (1986). The genetics of baculoviruses. In The Biology of Baculoviruses , pp. 217-238. Edited by R. R. Granados & B. A. Federici. Florida:CRC Press.

Nielsen, L. K., Smyth, G. K. & Greenfield, P. F. (1991). Hemocytometer cell count distributions: implications of non-Poisson behavior. Biotechnology Progress 7, 560-563.

Potter, K. N., Faulkner, P. & MacKinnon, E. A. (1976). Strain selection during serial passage of Trichoplusia ni nuclear polyhedrosis virus. Journal of Virology 18, 1040-1050.[Medline]

Potter, K. N., Jaques, R. P. & Faulkner, P. (1978). Modification of Trichoplusia ni nuclear polyhedrosis virus passaged in vivo. Intervirology 9, 76-85.[Medline]

Ramoska, W. A. & Hink, W. F. (1974). Electron microscope examination of two plaque variants from a nuclear polyhedrosis virus of the alfalfa looper, Autographa californica. Journal of Invertebrate Pathology 23, 197-201.[Medline]

Rhodes, D. J. (1996). Economics of baculovirus–insect cell production systems. Cytotechnology 20, 291-297.

Russell, R. L. Q. & Rohrmann, G. F. (1993). A 25 kDa protein is associated with the envelopes of occluded baculovirus virions. Journal of General Virology 195, 532-540.

Slavicek, J. M., Podgwaite, J. & Lanner-Herrera, C. (1992). Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from microbial pesticide Gypchek. Journal of Invertebrate Pathology 59, 142-148.

Slavicek, J. M., Hayes-Plazolles, N. & Kelly, M. E. (1995). Rapid formation of few polyhedra mutants of Lymantria dispar multinucleocapsid nuclear polyhedrosis virus during serial passage in cell culture. Biological Control 5, 251-261.

Sohi, S. S., Percy, J., Arif, B. M. & Cunningham, J. C. (1984). Replication and serial passage of a singly enveloped baculovirus of Orgyia leucostigma in homologous cell lines. Intervirology 21, 50-60.[Medline]

Taylor, J. R. (1997). The binomial distribution. In An Introduction to Error Analysis: The Study of Uncertainties in Physical Measurements, 2nd edn, pp. 227–244. California: University Science Books.

Wang, H. H., Fraser, M. J. & Cary, L. C. (1989). Transposon mutagenesis of baculoviruses: analysis of TFP3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis virus. Gene 81, 97-108.[Medline]

Wood, H. A. (1980). Isolation and replication of an occlusion body-deficient mutant of the Autographa californica nuclear polyhedrosis virus. Virology 105, 338-344.

Yamada, K., Sherman, K. E. & Maramorosch, K. (1982). Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line. Journal of Invertebrate Pathology 39, 185-191.

Received 6 November 2001; accepted 20 December 2001.