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Accumulation of few-polyhedra mutants upon serial passage of Anticarsia gemmatalis multiple nucleopolyhedrovirus in cell culture
Journal of Invertebrate Pathology 100 (2009) 153–159
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Accumulation of few-polyhedra mutants upon serial passage of Anticarsia gemmatalis multiple nucleopolyhedrovirus in cell culture Syomara Hakiko Matusita Soares de Rezende a,b, Maria Elita B. Castro a, Marlinda L. Souza a,* a b
Núcleo Temático de Controle Biológico – Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica W5 Norte Final, 70770-900 Brasília-DF, Brazil Departamento de Biologia Celular – Universidade de Brasília, Brasília-DF, Brazil
a r t i c l e
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Article history: Received 9 August 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Baculovirus In vitro production Serial passage Few-polyhedra mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus AgMNPV-2D Anticarsia gemmatalis
a b s t r a c t Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. To date, AgMNPV has been produced by larval infection and, due to in vivo production limitations and the continuing high demand for the biopesticide, attempts should be made to develop in vitro production of this virus. In order to investigate the effects caused by serial passage of AgMNPV in cell culture, we carried out a total of ten passages and analyzed the morphological and the genomic changes of the virus. After six passages, the many-polyhedra (MP) phenotype started to switch to the few-polyhedra (FP) phenotype which rapidly accumulated in the virus population. Ultrastructural analysis showed typical signs of FP mutant formation such as decrease in the number of polyhedra per cell, polyhedra aberrant morphology and low numbers of virions occluded in the protein matrix. Also enhanced BV production was observed from the fifth passage indicating that FP mutants were becoming predominant in comparison to the wild type virus. Restriction endonuclease analysis of the viral DNA revealed that lower and higher passages had similar profiles indicating that there were no large insertions or deletions or rearrangements in their genomes and indicating the generation of FP mutants instead of defective interfering viruses. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) belongs to the family Baculoviridae, classified in the genus Nucleopolyhedrovirus – NPV, Group I (Zanotto et al., 1993; Herniou et al., 2003; Theilmann et al., 2005) and is highly pathogenic for the velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae). This baculovirus has been widely used as a biopesticide in programs of biological control in Brazil (Moscardi, 1999; Moscardi and Souza, 2002). In spite of that, AgMNPV is still little understood at the molecular level and in its interactions with its host. The genome of Anticarsia gemmatalis MNPV isolate 2D (AgMNPV-2D) was recently sequenced and has a circular dsDNA genome (size of 132,239 bp) which is capable of encoding 152 non-overlapping open reading frames (ORFs) (Oliveira et al., 2006). Currently, approximately 1.2 million hectares of soybean are treated with AgMNPV which has been produced by host infection. However, due to limitations of in vivo production and considering technical issues, the development of cell culture technology for AgMNPV production would be very attractive. Baculoviruses produce occluded virus in a protein matrix called polyhedra, which allows the virus to survive in the environment and transmit the disease from one insect to another. A second form * Corresponding author. Fax: +55 61 3448 4673. E-mail address: [email protected] (M.L. Souza). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.12.002
of the virus is produced during the infection, a budded virus particle (BV), which serves for the transmission of the virus to other tissues of the caterpillar body and is also the form used for inoculation in cell culture. The establishment of insect cell lines permissive to viral replication has permitted the purification of viral clones by plaque assays, and promoted the advance of the molecular biology of baculoviruses. However, studies have shown frequent accumulation of spontaneous deletions during serial virus passage in cell culture (reviewed by Krell, 1996). Upon serial passage, two main classes of virus may arise at a high frequency and becomes predominant, few-polyhedra (FP) mutants and the Defective interfering virus (DIs). The latter have a smaller genome size and require a standard helper virus for the initiation of infection (Kool et al., 1991; Lee and Krell, 1992; Pijlman et al., 2001). DIs are the most prevalent when infections started at high multiplicities of infection since there is a higher probability of these particles to co-infect cells with the helper virus (Wickham et al., 1991). FP mutants have been reported in several baculoviruses including Autographa californica MNPV (Hink and Vail, 1973), Trichoplusia ni MNPV (Potter et al., 1976), Galleria mellonella MNPV (Fraser et al., 1983), Lymantria dispar MNPV (Slavicek et al., 1992), Orgyia pseudotsugata MNPV (Russell and Rohrmann, 1993), Helicoverpa armigera SNPV (Chakraborty and Reid, 1999; Lua et al., 2002) and Spodoptera frugiperda MNPV(Pedrini et al., 2004).
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Mutations in the 25 kDa protein gene are involved in generation of the FP phenotype and result in reduced virion occlusion, altered intranuclear envelopment and enhanced BV production (Fraser et al., 1983, 1985; Beames and Summers, 1988, 1989; Harrison and Summers, 1995; Slavicek et al., 1995). Recently, Wu et al. (2005) constructed a fp25K null mutant based on a polyhedrin-null bacmid system to study the function of the FP25K of Helicoverpa armigera SNPV. Their results confirmed, by rescue experiments, that the deletion of fp25k caused a higher BV yield and a decrease in progeny occlusion derived virus (ODVs) progeny. A strain of LdMNPV that did not accumulate FP mutants after extended serial passage in cell culture was characterized by Slavicek et al. (2001). The isolate produced more BV than wild-type virus and essentially the same amount of BV as FP mutant. Similarly, Pedrini et al. (2005) described the isolation of a unique HaSNPV mutant that exhibited a partial many polyhedra (MP) and few polyhedra (FP) phenotype and produced the same amount of BV as the FP mutant. Although, the yield of polyhedra of these stable MP variants is lower that the wild-type, they are competitive with FP mutants and could contribute to overcome limitations on in vitro production. In this study, AgMNPV-2D was serially passaged in cell culture until predominance of the FP phenotype was reached. A total of ten passages was carried out and the few polyhedra phenotype started to accumulate from passage six onwards. While the amount of polyhedra was reduced, the number of BV increased and abnormal polyhedra started to be formed. REN Analysis of the genomic DNA did not reveal any significant alteration indicating that small changes at 25fpk locus, such as single nucleotide insertions or deletions, are leading to accumulation of FP mutants in the viral population.
with FP), and hypertrophied but without polyhedra in the nuclei were determined using a Neubauer haemocytometer. The total number of polyhedra per cell was determined based on the number of cells possessing polyhedra and number of polyhedral inclusion bodies per ml (PIBs/ml). The last one (polyhedra density) was calculated after lysis of infected cells with 1% sodium dodecyl sulphate at room temperature for l h. Infected cells at 72 h p.i. were pelleted by centrifugation at 3000 rpm in a clinical centrifuge for 5 min. The supernatant containing budded virus (1 ml) was used as viral inoculum (P1) for a new passage, at a density of 2 106 cells in 25 T cell culture flask, and the pellet was separated for electron microscopy analysis. In the same way, subsequent passages always used 1 ml of the previous supernatant. Triplicate flasks of 2 106 infected cells were done for every passage. Typical cells of each serial passage were photographed using a Zeiss Axiovert 135 M microscope. 2.4. Budded virus titration The BV production was determined by the end-point dilution assay (O’Reilly et al., 1992). BTI-Tn5B1-4 cells were seeded in 96 wells plate (1 104 per well), infected with different serial dilutions of the virus (10 1–10 10) and incubated at 27 °C. Cells were monitored daily. At 5 days after infection, wells containing cells with polyhedra, or typical signs of baculovirus infection such as cell rounding and nuclear hypertrophy, were counted as positive. The viral titer was established using the method of Reed and Muench (1938). 2.5. Transmission electron microscopy
T. ni cells, BTI-Tn-5B1-4 (Granados et al., 1994), known as ‘‘High five” cells, were grown as monolayers in TMNFH medium supplemented with 10% fetal bovine serum, at 27 °C. The AgMNPV strain 2D (Johnson and Maruniak, 1989), a plaque purified virus, was serially passed in culture cells. A. gemmatalis larvae were obtained from the Insect Rearing Laboratory of EMBRAPA-CENARGEN (Brasília, Brazil).
Pellets containing infected cells from passages P2, P4, P6, P8 and P10 were suspended in 2.5% glutaraldehyde fixative and 0.1 M sodium cacodylate buffer, pH 7.2 and kept at 4 °C for 24 h. The pellets were washed 3 in 0.1 M sodium cacodylate buffer, pH 7.2 and post-fixed with 2% osmium tetroxide in the same buffer. After dehydration in a standard ethanol series the samples were embedded in Spurr resin. Ultra fine sections were obtained with an ultramicrotome equipped with a diamond knife and mounted on copper grids. Sections were contrasted with 3% uranyl acetate and viewed on a Jeol 1011 microscope. The relative diameters of polyhedra of some samples were determined by measurement of the diameter of polyhedra cross sections in electron micrographs.
2.2. In vitro initial passaging of AgMNPV-2D
2.6. Restriction endonuclease analysis of virus DNA
Fourth instar A. gemmatalis larvae were per os infected with AgMNPV-2D by placing them on a diet containing surface-applied polyhedra (1 106 per diet surface). The hemolymph was collected on the 4th day post-infection (d.p.i), by larvae bleed and cysteine was added at 10 mM. Hemolymph was then passed through a syringe filter (0.45 lm) for sterilization. This solution was used as inoculum (passage 0) for three individual infections on Tn5B1-4 cells plated at a density of 2 106 cells/flask in 25 T cell culture flasks. At the end of the incubation period (1 h, 27 °C) the inoculum was removed. The cells were then rinsed with serum free TNMFH medium and maintained in complete medium at 27 °C. This infection constituted the first passage (P1) of virus in cell culture.
Budded virus from passages P1, P2, P4, P6 and P8 (supernatant) were purified in a 25% sucrose cushion by ultracentrifugation in a Sorvall SW28 rotor at 24,000 rpm, for 75 min at 4 °C (O’Reilly et al., 1992). For DNA extraction the pellet was suspended in 500 ll of disruption buffer (10 mM Tris, pH 7.6; 10 mM EDTA, pH 8.0; 0.25% SDS), containing 500 lg ml 1 of Proteinase K, and the sample was incubated overnight at 37 °C. DNA was extracted with phenol:chloroform and then digested with HindIII or BamHI enzymes, according to the manufacture (Promega) at 37 °C for 3 h. The digested DNA samples were loaded into a 0.8% agarose gel, containing ethidium bromide (0.3 lg/ml) and electrophoresed at 40 V.
2. Material and methods 2.1. Cell lines, viruses and insects
3. Results 2.3. Serial passage of BV 3.1. In vitro serial passage of AgMNPV-2D Three aliquots of cell suspension from each flask were collected at 72 h.p.i., and were visualized by phase contrast microscopy (Olympus CK2). The percentages of cells with many polyhedra in the nuclei (cells with MP), with few polyhedra in the nuclei (cells
The effect of serial passage of AgMNPV-2D in T. ni cells cultures was initially investigated through analysis of the percentage of cells showing occlusion bodies and the number of polyhedra pro-
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duced per cell. Ten passages of the virus in BTI-Tn5B1-4 cells were performed, and the infected cells were observed up to 72 h p.i. under a phase contrast microscope. A decrease in cell percentage with many polyhedra (MP infected cells) yield was noted as the AgMNPV-2D was serially passaged (Fig. 1). The drop was most pronounced from passage 5 to passage 6. During passaging, the percentage of cells with MP decreased while there was an increase in the percentage of cells with few polyhedra (FP infected cells). However, there was a slight increase in MP production from passage 6 to passage 8 after an initial fall. The steep slope between passage 5 and passage 6 indicates a sharp transition from an MP dominated population to a FP one. These decreases of cells with MP and increase of cells with FP is a feature of FP mutation common in prolonged viral infections in cell culture. The experiments were done by infection of 2 106 cells in 25 T flask using a fixed volume of virus (1 ml). The correlation between the virus titer obtained upon serial passage (Table 1) in those cells show a great range of the MOI. Up to the fourth passage this would represent a MOI of approximately 4. However, by the sixth passage it increases dramatically to about 1 104 viruses/cell. The polyhedra production also decreased during passage (Fig. 2). Initially, from passage 1 to passage 2, there was an increase in the number of polyhedra formed. However, after this passage the number of occlusion bodies decreased dramatically to very few polyhedra. Again the greatest decrease occurred from passages 5 to passage 6. On the other hand, BV production increased with higher passage number, reaching 1.1 1010 and 1.5 1010 PFU ml 1 at passages 5 and 7, corresponding with a gradual shift of phenotype from MP to FP phenotype. The BV titers declined at very later passage (P9) when the phenotype should have been overwhelmingly FP (Table 1).
Table 1 Budded virus production during AgMNPV-2D serial passage in cell culture. Passage number PI P3 P5 P7 P9
Virus titer(PFU/ml) 6
7.1 10 8.0 106 1.1 1010 1.5 1010 3.8 108
SD ±5.21 106 ±4.24 106 ±1.08 1010 ±8.38 109 ±1.43 108
The virus titers were determined by the 50% tissue culture infective dose (TCID50) method. The values are the averages of three determinations. The standard deviation (SD) is shown for each value.
3.2. Cell morphology alterations during virus serial passage The cell morphology of MP and FP infected cells at each passage was analyzed using phase contrast microscopy. Signs of infection,
Fig. 1. Percentage of cells that contain polyhedra during serial passage of AgMNPV2D. The percentage of cells with many polyhedra (MP) and with few polyhedra (FP) was determined after each serial passage of the virus. The values are the averages of three determinations. The vertical lines indicate the standard deviation.
Fig. 2. Polyhedra production during serial passage of AgMNPV-2D. The number of polyhedra produced per cell, within cells that produced polyhedra, was determined after each serial passage in cell culture. The values shown are the averages of three determinations. The vertical lines indicate the standard deviation.
such as larger cells and nuclear hypertrophy, presence of virogenic stroma and protrusions were observed in all cells. However, formation of occlusion bodies was not seen in all of them and was very rare in later passages. Mock infected cells were used as control (Fig. 3a.). Infected cells from passages P1, P3, P6, P8 and P10 are shown in Fig. 3 (b–f, respectively). In the initial passages (P1 and P3) some cytopathic effects as cell rounding, nuclear hypertrophy and formation of polyhedra were evident and high number of cells with MP (Fig. 3b and c) was observed. An increase in the rate of FP and reduction in MP was noted at the sixth passage (Fig. 3d). After this passage, the infected cells FP appear in lower number but appear larger. In latter passages (P8 and P10) the majority of cells are FP cells (Fig. 3e and f). The ultrastructure of infected cells was visualized by transmission electron microscopy. The cytopathic effects in infected BTI-Tn5B14 cells resulting from passages P2, P4, P6, P8 and P10 are shown in Fig. 4 (A–E, respectively). In initials passages the cells showed nuclear and cell hypertrophy with formation of many polyhedra (Fig. 4A and B) and the presence of numerous occluded virions within them (Fig. 5A). Upon serial passage the cells displayed a reduced number of polyhedra (Fig. 4C) or even had no polyhedra (Fig. 4D and E). In latter passages, as the number of polyhedra decreased the virogenic stroma became more evident with production of many virions (Fig. 4E and F).
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Fig. 3. Trichoplusia ni BTI-Tn5B1-4 cells infected with AgMNPV-2D, 72 h p.i. Phase contrast micrographs (a) Mock infected cells; passages: (b) P1, (c) P3, (d) P6, (e) P8, (f) P10. Cells were viewed with an Axiovert 135 M inverted microscope at 200 (a, d, f) and 320 (b, c, e) magnification.
The morphology of the occlusion bodies revealed that in later passages polyhedra usually have little or no virions occluded and may have aberrant morphology (Fig. 5B–D). In contrast, polyhedra with many virions (Fig. 5A) were produced at the initial passage. In addition, measurements of the relative polyhedra diameter showed that they ranged in size from 1.0 to 2.0 lm, with an average size of 1.4 ± 0.05 lm up to fifth passage and of 1.7 ± 0.05 lm at late passages. The decrease in numbers of occlusion bodies during passage in parallel to the increase of their sizes were visualized in most cells. 3.3. Restriction endonuclease analysis of virus DNA The HindIII digests of virus DNA from passages P1, P2, P4, P6 and P8 DNA were carried out to determine if gross genomic changes or rearrangements had occurred during serial passage and if this could be related to the appearance of the mutants. Comparison of the digest pattern from the first passage (P1) with the
subsequent ones showed no differences in fragment sizes (Fig. 6). No large insertions or deletions were detected. The same results were found upon digestion with BamHI (data not shown). 4. Discussion The serial passage of AgMNPV in BTI-Tn5B1-4 cells leads to accumulation of the FP phenotype. This effect has been reported for NPV groups I and II and is associated with generation of two main type of mutations, the ones termed FP mutants and the defective interfering viruses (DIs) (reviewed by Krell, 1996). In this work, the virus population clearly switched from the wild-type many polyhedra (MP) per cell phenotype to the few polyhedra (FP) per cell phenotype (Fig. 1). The FP phenotype started to accumulate on passage 6 and became predominant to the latest passage (P10). Rapid accumulation of FP mutants in cell culture has also been described for other baculovirus such as LdMNPV (Slavicek
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Fig. 5. Electron micrographs showing polyhedra and their occluded virions. (A) typical polyhedra (passage 2) with many occluded virions. (B) and (C) Polyhedra with few occluded virions (passage 8). (D) Polyhedra with aberrant morphology (passage 10).
bp 12,216 11,198 10,180 9,162 8,144 7,126 6,108
Fig. 4. Electron micrographs showing characteristics of many polyhedra phenotype and few polyhedra phenotype of AgMNPV infected cells. (A) and (B) cell with many polyhedra in the nucleus. (C) cell with few polyhedra. (D) cell with none polyhedra. (E) cell showing the virogenic stroma. (F) Detail of virogenic stroma with many virions. Panels A–F represent passage 2, 4, 6, 8, 10 and 10, respectively.
et al., 1995, 1996, 2001), HaSNPV (Lua et al., 2002) and SfMNPV (Pedrini et al., 2004). There was a tremendous 30-fold decrease in the yield of polyhedra during sequential passage and reached a phase in which occluded virus were very rare in cells (Figs. 2 and 3). This leads to a strong decrease of virulence since production of occlusion bodies is the only form of transmission of the disease in nature (Padmavathamma and Veeresh, 1991). Simultaneously, there was an increase in the yield of budded virus particles (Table 1). The stability and selective advantage of the FP mutant on serial passage are attributed to its enhanced BV production (Potter et al., 1976; Fraser and Hink, 1982; Fraser and McCarthy, 1984). FP mutants are often selected for in cell culture because they have rates of BV replication greater than those displayed by the wild-type (Harrison and Summers, 1995). The ultrastructural changes shown in infected cells serially passed with AgMNPV are typical of FP mutants. Not only the cells exhibited few polyhedra with aberrant shape, but there was minimal virion occlusion in these bodies (Figs. 4 and 5). At higher passages, instead of virus occlusion, many cells presented a well defined virogenic stroma region containing many virions, indicating intensive replication of the virus (Fig. 4E and F). Besides, polyhedra appears to grow larger during passages. These traits are the same as those exhibited by AcMNPV (Wood, 1980; Harrison and
5,090
M1
P1
P2
P4
P6
P8
M2
bp 11,500
5,000 4,600 4,500
4,072 3,054
2,800 2,600
2,036
2,100 1,900 1,700
1,636 1,100 1,000 1,018 506
800
396 344 298 220 201 154
Fig. 6. Restriction endonuclease analysis of AgMNPV-2D DNA from passages P1, P2, P4, P6 and P8. Viral genomic DNA was digested with HindIII. The fragments were separated by 0.8% agarose gel electrophoresis, and the gel was stained with ethidium bromide. Markers in base pairs: (M1) 1kb DNA ladder and (M2) DNA lambda/PstI.
Summers, 1995), TnMNPV (Potter et al., 1976), GmMNPV (Fraser and Hink, 1982), LdMNPV (Slavicek et al., 1995), HaSNPV (Lua
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et al., 2002) and SfMNPV (Pedrini et al., 2004). The polyhedra of FP mutants are reported to have much lower biological activity in in vivo assays (Potter et al., 1976; Fraser and Hink, 1982; Slavicek et al., 1995, Lua et al., 2002). Genomic restriction endonuclease digestion profiles of AgMNPV DNA from all passages were similar with no evidence of large insertions, deletions or rearrangements (Fig. 6). The fact that no significant changes were detected by REN analysis suggests that small changes such as point mutations, single nucleotide insertions or deletions, might be occurring in the AgMNPV 25kfp gene leading to FP mutants generation. However, we cannot run out the possibility that defective interfering viruses might have also been produced, at least at later passages. DIs and FP mutants induce the same cytopathic effects and similar polyhedra yield and morphology. Therefore DI particles may have contributed to some of the CPE observed. At later passages, when there was a dramatic increase in the MOI instead of only FP mutants, DIs could have been also developed which may not be evidenced at the DNA restriction profile. In addition, the up to 40-fold drop in BV production from passage 7 to passage 9 (Table 1) might have been due to DIs. In a previous study the biological constraints and bioprocess issues for in vitro production of AgMNPV was investigated (Rodas et al., 2005). For serial passage of the virus, SF9 cells (1 106 ml) were cultivated in Schott–shaker system (20 ml) and infected with a wild-type AgMNPV (not plaque purified) at MOI of 1. Analysis of the restriction profiles of viral DNA digested with HindIII and PstI enzymes showed that defective interfering particles (DIs) were formed during passaging of the virus in cell culture. However, the specific conditions used in our experiments were different. For instance, we used a different cell line (BTI-Tn5B1-4) and a plaque purified virus as inoculum (AgMNPV-2D), instead of a field isolate. It has been recently reported that while BTI-Tn5B1-4 cells are very productive for AgMNPV, the SF9 cells are much less susceptible to this virus with lower yields of BV and occlusion bodies (Castro et al., 2006). The appearance of the FP phenotype during serial passage in cell culture often correlates with the presence of DNA insertions and deletions and have been reported for AcMNPV and GmMNPV (Fraser et al., 1983; Kumar and Miller, 1987; Cary et al., 1989; Bull et al., 2003) that occur predominantly in the 25 K locus (Beames and Summers, 1989). Spontaneous insertion of transposable elements originating from host cell DNA into the viral 25kfp gene has been shown to be a common cause of the FP phenotype (Cary et al., 1989; Bull et al., 2003). In contrast to the other baculovirus FP studied, most LdMNPV FP mutants do not contain large insertions or deletions in the 25kfp gene (Slavicek et al., 1995). The majority of the FP mutants of LdMNPV contained single nucleotide insertions or small deletions (Bischoff and Slavicek, 1997). The same was described for HaSNPV (Lua et al., 2002) during passaging of this virus in cell culture. The sequences obtained from individual clones of the 25kfp gene PCR products of a late passage revealed point mutations occurring during throughout the gene. Our results seem to be similar to the ones obtained with LdMNPV and HaSNPV FP mutants. The feasibility of in vitro AgMNPV production depends on overcoming the limitations caused by virus passaging. Based in our studies the number of times that AgMNPV is passaged in cell culture should be kept to a minimum to minimize the generation of FP mutants. Therefore, preparation of large low-passage stocks is recommend. While Tn5B1-4 cells may be appropriate for production of AgMNPV producers should take care to not passage the virus more than five times. In addition, for large scale virus production a low MOI infection is required to prevent formation of DI particles.
The generation of FP mutants is a limitation for successful production of polyhedra in cell culture bioreactors. In further studies, we intend to sequence of the 25kfp gene of several FP plaque purified clones in order to characterize the most frequent mutations caused by serial passage of AgMNPV in cell culture. Also the selection of stable many polyhedra (MP) variants, competitive to FP mutants and more resistant to the passage effect, is another strategy that will be applied to the development of AgMNPV in vitro production. Acknowledgments The work is supported by grants from Embrapa and CNPq. The authors would like to thank Dr. Antonio Williams Moita, from Embrapa Hortaliças, for the statistical analysis and Dr. Bergmann Morais Ribeiro, from University of Brasilia, for electron microscope support. We also thank Dr. Peter Ward, Inglis (Embrapa Recursos Genéticos e Biotecnologia) for scientific editing of the manuscript. References Beames, B., Summers, M.D., 1988. Comparison of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra baculovirus mutants. Virology 162, 206–220. Beames, B., Summers, M.D., 1989. Location and nucleotide sequence of the 25 K protein missing from baculovirus few polyhedra (FP) mutants. Virology 168, 344–353. Bischoff, D.S., Slavicek, J.M., 1997. Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25KFP gene may be caused by DNA replication errors. J. Virol. 71, 1097–1106. Bull, J.C., Godfray, H.C., O’Reilly, D.R., 2003. A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni. Appl. Environ. Microbiol. 69, 2052–2057. Cary, L.C., Goebel, M., Corsaro, B.G., Wang, H.G., Rosen, E., Fraser, M.J., 1989. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156–169. Castro, M.E.B., Ribeiro, Z.M.A., Souza, M.L., 2006. Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: morphology, viral production and protein synthesis. Biol. Control 36, 299–304. Chakraborty, S., Reid, S., 1999. Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures. J. Gen. Virol. 73, 303– 308. Fraser, M.J., Hink, W.F., 1982. The isolation and characterization of the MP and FP plaque variants of Galleria mellonella nuclear polyhedrosis virus. Virology 117, 366–378. Fraser, M.J., McCarthy, W.J., 1984. The detection of FP plaque variants of Heliothis zea. J. Invertebr. Pathol. 43, 427–429. 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. J. Virol. 47, 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. Granados, R.R., Li, G., Derksen, A.C.G., McKenna, K.A., 1994. A new cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J. Invertebr. Pathol. 64, 260–266. Harrison, R.L., Summers, M.D., 1995. 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. J. Gen. Virol. 76, 1451–1459. Herniou, E.A., Olszewski, J.A., Cory, J.S., O’Reilly, D.R., 2003. The genome sequence and evolution of baculoviruses. Annu. Rev. Entomol. 48, 211–234. Hink, W.F., Vail, P.V., 1973. A plaque assay for titration of alfalfa lopper nuclear polyhedrosis virus in a cabbage lopper (TN 368) cell line. J. Invertebr. Pathol. 22, 168–174. Johnson, D.W., Maruniak, J.E., 1989. Physical map of Anticarsia gemmatalis nuclear polyhedrosis virus (AgMNPV-2) DNA. J. Gen. Virol. 70, 1877–1883. Kool, M., Voncken, J.W., van Lier, F.L., Tramper, J., Vlak, J.M., 1991. Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology 183, 739–746. Krell, P.J., 1996. Passage effect of virus infection in insect cells. Cytotechnology 20, 125–137. Kumar, S., Miller, L.K., 1987. Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture. Virus Res. 7, 335–349. Lee, H.Y., Krell, P.J., 1992. Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus. J. Virol. 66, 4339–4347.
S. H. M. S. d. Rezende et al. / Journal of Invertebrate Pathology 100 (2009) 153–159 Lua, L.H.L., Pedrini, M.R.S., Reid, S., Robertson, A., Tribe, D.E., 2002. Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. J. Gen. Virol. 83, 945–955. Moscardi, F., 1999. Assessment of the application of baculoviruses for control of lepidoptera. Ann. Rev. Entomol. 44, 257–289. Moscardi, F., Souza, M.L., 2002. Baculovírus para o controle de pragas: Panacéia ou realidade? Biotecnol. Ciênc. Des. 24, 22–29. Oliveira, J.V.C., Wolff, J.L.C., Garcia-Maruniak, A., Ribeiro, B.M., Castro, M.E.B., Souza, M.L., Moscardi, F., Maruniak, J.E., Zanotto, P.M.A., 2006. Genome of the most widely used viral biopesticide: Anticarsia gemmatalis multiple nucleopolyhedrovirus. J. Gen. Virol. 87, 3233–3250. O’Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus Expression Vectors: A Laboratory Manual. Oxford Univ. Press, New York. Padmavathamma, K., Veeresh, G.K., 1991. Effect of larval age and dosage of nuclear polyhedrosis virus on the susceptibility of diamondback moth, Plutella xylostella. Entomol. Exp. Appl. 60, 39–42. Pedrini, M.R.S., Wolff, J.L.C., Reid, S., 2004. Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures. Ann. Appl. Biol. 145, 107–112. Pedrini, M.R.S., Nielsen, L.K., Reid, S., Chan, L.C.L., 2005. 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. In vitro Cell. Dev. Biol. Animal 41, 289–297. Pijlman, G.P., Born, E.V.D., Martens, D.E., Vlak, J.M., 2001. Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283, 132–138. Potter, K.N., Faulkner, P., MacKinnon, E.A., 1976. Strain selection during serial passage of Trichoplusia ni nuclear polyhedrosis virus. J. Virol. 18, 1040–1050. Reed, L., Muench, H., 1938. A simple method for estimating fifty percent endpoints. Am. J. Hygenf. 27, 493–497. Rodas, V.M., Marques, F.H., Honda, M.T., Soares, D.M., Jorge, S.A.C., Antoniazzi, M.M., Medugno, C., Castro, M.E.B., Ribeiro, B.M., Souza, M.L., Tonso, A., Pereira, C.A.,
159
2005. Cell culture derived AgMNPV bioinsecticide: biological constraints and bioprocess issues. Cytotechnology 48, 27–39. Russell, R.L.Q., Rohrmann, G.F., 1993. A 25-kDa protein is associated with the envelopes of occluded baculovirus virions. Virology 195, 532–540. Slavicek, J.M., Podgewaite, J., Lanner-Herrera, C., 1992. Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide Gypchek. J. Invertebr. Pathol. 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. Biol. Control 5, 251–261. Slavicek, J.M., Mercer, M.J., Kelly, M.E., Hayes-Plazolles, N., 1996. Isolation of a baculovirus variant that exhibits enhanced polyhedra production stability during serial passage in cell culture. J. Invertebr. Pathol. 67, 153–160. Slavicek, J.M., Hayes-Plazolles, N., Kelly, M.E., 2001. Identification of a Lymantria dispar nucleopolyhedrovirus isolate that does not accumulate few polyhedra mutants during extended serial passage in cell culture. Biol. Control 22, 159– 168. Theilmann, D.A., Blissard, G.W., Bonning, B., Jehle, J., O´Reilly, D.R., Rohrmann, G.F., Thiem, S., Vlak, J.M., 2005. Baculoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Eighth Report of the International Committee on Taxonomy of Viruses. Eselvier Academic Press, Amsterdam, pp. 177–185. Wickham, T.J., Davis, T., Granados, R.R., Hammer, D.A., Shuler, M.L., Wood, H.A., 1991. Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection. Biotechnol. Lett. 13, 483–488. Wood, H.A., 1980. Isolation and replication of an occlusion body-deficient mutant of the Autographa californica nuclear polyhedrosis virus. Virology 105, 338–344. Wu, D., Deng, F., Sun, X., Wang, H., Yuan, L., Vlak, J.M., Hu, Z., 2005. Functional analysis of FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. J. Gen. Virol. 86, 2439–2444. Zanotto, P.M., Kessing, B.D., Maruniak, J.E., 1993. Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J. Invertebr. Pathol. 62, 147–164.
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Accumulation of few-polyhedra mutants upon serial passage of Anticarsia gemmatalis multiple nucleopolyhedrovirus in cell culture Syomara Hakiko Matusita Soares de Rezende a,b, Maria Elita B. Castro a, Marlinda L. Souza a,* a b
Núcleo Temático de Controle Biológico – Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica W5 Norte Final, 70770-900 Brasília-DF, Brazil Departamento de Biologia Celular – Universidade de Brasília, Brasília-DF, Brazil
a r t i c l e
i n f o
Article history: Received 9 August 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Baculovirus In vitro production Serial passage Few-polyhedra mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus AgMNPV-2D Anticarsia gemmatalis
a b s t r a c t Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. To date, AgMNPV has been produced by larval infection and, due to in vivo production limitations and the continuing high demand for the biopesticide, attempts should be made to develop in vitro production of this virus. In order to investigate the effects caused by serial passage of AgMNPV in cell culture, we carried out a total of ten passages and analyzed the morphological and the genomic changes of the virus. After six passages, the many-polyhedra (MP) phenotype started to switch to the few-polyhedra (FP) phenotype which rapidly accumulated in the virus population. Ultrastructural analysis showed typical signs of FP mutant formation such as decrease in the number of polyhedra per cell, polyhedra aberrant morphology and low numbers of virions occluded in the protein matrix. Also enhanced BV production was observed from the fifth passage indicating that FP mutants were becoming predominant in comparison to the wild type virus. Restriction endonuclease analysis of the viral DNA revealed that lower and higher passages had similar profiles indicating that there were no large insertions or deletions or rearrangements in their genomes and indicating the generation of FP mutants instead of defective interfering viruses. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) belongs to the family Baculoviridae, classified in the genus Nucleopolyhedrovirus – NPV, Group I (Zanotto et al., 1993; Herniou et al., 2003; Theilmann et al., 2005) and is highly pathogenic for the velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae). This baculovirus has been widely used as a biopesticide in programs of biological control in Brazil (Moscardi, 1999; Moscardi and Souza, 2002). In spite of that, AgMNPV is still little understood at the molecular level and in its interactions with its host. The genome of Anticarsia gemmatalis MNPV isolate 2D (AgMNPV-2D) was recently sequenced and has a circular dsDNA genome (size of 132,239 bp) which is capable of encoding 152 non-overlapping open reading frames (ORFs) (Oliveira et al., 2006). Currently, approximately 1.2 million hectares of soybean are treated with AgMNPV which has been produced by host infection. However, due to limitations of in vivo production and considering technical issues, the development of cell culture technology for AgMNPV production would be very attractive. Baculoviruses produce occluded virus in a protein matrix called polyhedra, which allows the virus to survive in the environment and transmit the disease from one insect to another. A second form * Corresponding author. Fax: +55 61 3448 4673. E-mail address: [email protected] (M.L. Souza). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.12.002
of the virus is produced during the infection, a budded virus particle (BV), which serves for the transmission of the virus to other tissues of the caterpillar body and is also the form used for inoculation in cell culture. The establishment of insect cell lines permissive to viral replication has permitted the purification of viral clones by plaque assays, and promoted the advance of the molecular biology of baculoviruses. However, studies have shown frequent accumulation of spontaneous deletions during serial virus passage in cell culture (reviewed by Krell, 1996). Upon serial passage, two main classes of virus may arise at a high frequency and becomes predominant, few-polyhedra (FP) mutants and the Defective interfering virus (DIs). The latter have a smaller genome size and require a standard helper virus for the initiation of infection (Kool et al., 1991; Lee and Krell, 1992; Pijlman et al., 2001). DIs are the most prevalent when infections started at high multiplicities of infection since there is a higher probability of these particles to co-infect cells with the helper virus (Wickham et al., 1991). FP mutants have been reported in several baculoviruses including Autographa californica MNPV (Hink and Vail, 1973), Trichoplusia ni MNPV (Potter et al., 1976), Galleria mellonella MNPV (Fraser et al., 1983), Lymantria dispar MNPV (Slavicek et al., 1992), Orgyia pseudotsugata MNPV (Russell and Rohrmann, 1993), Helicoverpa armigera SNPV (Chakraborty and Reid, 1999; Lua et al., 2002) and Spodoptera frugiperda MNPV(Pedrini et al., 2004).
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Mutations in the 25 kDa protein gene are involved in generation of the FP phenotype and result in reduced virion occlusion, altered intranuclear envelopment and enhanced BV production (Fraser et al., 1983, 1985; Beames and Summers, 1988, 1989; Harrison and Summers, 1995; Slavicek et al., 1995). Recently, Wu et al. (2005) constructed a fp25K null mutant based on a polyhedrin-null bacmid system to study the function of the FP25K of Helicoverpa armigera SNPV. Their results confirmed, by rescue experiments, that the deletion of fp25k caused a higher BV yield and a decrease in progeny occlusion derived virus (ODVs) progeny. A strain of LdMNPV that did not accumulate FP mutants after extended serial passage in cell culture was characterized by Slavicek et al. (2001). The isolate produced more BV than wild-type virus and essentially the same amount of BV as FP mutant. Similarly, Pedrini et al. (2005) described the isolation of a unique HaSNPV mutant that exhibited a partial many polyhedra (MP) and few polyhedra (FP) phenotype and produced the same amount of BV as the FP mutant. Although, the yield of polyhedra of these stable MP variants is lower that the wild-type, they are competitive with FP mutants and could contribute to overcome limitations on in vitro production. In this study, AgMNPV-2D was serially passaged in cell culture until predominance of the FP phenotype was reached. A total of ten passages was carried out and the few polyhedra phenotype started to accumulate from passage six onwards. While the amount of polyhedra was reduced, the number of BV increased and abnormal polyhedra started to be formed. REN Analysis of the genomic DNA did not reveal any significant alteration indicating that small changes at 25fpk locus, such as single nucleotide insertions or deletions, are leading to accumulation of FP mutants in the viral population.
with FP), and hypertrophied but without polyhedra in the nuclei were determined using a Neubauer haemocytometer. The total number of polyhedra per cell was determined based on the number of cells possessing polyhedra and number of polyhedral inclusion bodies per ml (PIBs/ml). The last one (polyhedra density) was calculated after lysis of infected cells with 1% sodium dodecyl sulphate at room temperature for l h. Infected cells at 72 h p.i. were pelleted by centrifugation at 3000 rpm in a clinical centrifuge for 5 min. The supernatant containing budded virus (1 ml) was used as viral inoculum (P1) for a new passage, at a density of 2 106 cells in 25 T cell culture flask, and the pellet was separated for electron microscopy analysis. In the same way, subsequent passages always used 1 ml of the previous supernatant. Triplicate flasks of 2 106 infected cells were done for every passage. Typical cells of each serial passage were photographed using a Zeiss Axiovert 135 M microscope. 2.4. Budded virus titration The BV production was determined by the end-point dilution assay (O’Reilly et al., 1992). BTI-Tn5B1-4 cells were seeded in 96 wells plate (1 104 per well), infected with different serial dilutions of the virus (10 1–10 10) and incubated at 27 °C. Cells were monitored daily. At 5 days after infection, wells containing cells with polyhedra, or typical signs of baculovirus infection such as cell rounding and nuclear hypertrophy, were counted as positive. The viral titer was established using the method of Reed and Muench (1938). 2.5. Transmission electron microscopy
T. ni cells, BTI-Tn-5B1-4 (Granados et al., 1994), known as ‘‘High five” cells, were grown as monolayers in TMNFH medium supplemented with 10% fetal bovine serum, at 27 °C. The AgMNPV strain 2D (Johnson and Maruniak, 1989), a plaque purified virus, was serially passed in culture cells. A. gemmatalis larvae were obtained from the Insect Rearing Laboratory of EMBRAPA-CENARGEN (Brasília, Brazil).
Pellets containing infected cells from passages P2, P4, P6, P8 and P10 were suspended in 2.5% glutaraldehyde fixative and 0.1 M sodium cacodylate buffer, pH 7.2 and kept at 4 °C for 24 h. The pellets were washed 3 in 0.1 M sodium cacodylate buffer, pH 7.2 and post-fixed with 2% osmium tetroxide in the same buffer. After dehydration in a standard ethanol series the samples were embedded in Spurr resin. Ultra fine sections were obtained with an ultramicrotome equipped with a diamond knife and mounted on copper grids. Sections were contrasted with 3% uranyl acetate and viewed on a Jeol 1011 microscope. The relative diameters of polyhedra of some samples were determined by measurement of the diameter of polyhedra cross sections in electron micrographs.
2.2. In vitro initial passaging of AgMNPV-2D
2.6. Restriction endonuclease analysis of virus DNA
Fourth instar A. gemmatalis larvae were per os infected with AgMNPV-2D by placing them on a diet containing surface-applied polyhedra (1 106 per diet surface). The hemolymph was collected on the 4th day post-infection (d.p.i), by larvae bleed and cysteine was added at 10 mM. Hemolymph was then passed through a syringe filter (0.45 lm) for sterilization. This solution was used as inoculum (passage 0) for three individual infections on Tn5B1-4 cells plated at a density of 2 106 cells/flask in 25 T cell culture flasks. At the end of the incubation period (1 h, 27 °C) the inoculum was removed. The cells were then rinsed with serum free TNMFH medium and maintained in complete medium at 27 °C. This infection constituted the first passage (P1) of virus in cell culture.
Budded virus from passages P1, P2, P4, P6 and P8 (supernatant) were purified in a 25% sucrose cushion by ultracentrifugation in a Sorvall SW28 rotor at 24,000 rpm, for 75 min at 4 °C (O’Reilly et al., 1992). For DNA extraction the pellet was suspended in 500 ll of disruption buffer (10 mM Tris, pH 7.6; 10 mM EDTA, pH 8.0; 0.25% SDS), containing 500 lg ml 1 of Proteinase K, and the sample was incubated overnight at 37 °C. DNA was extracted with phenol:chloroform and then digested with HindIII or BamHI enzymes, according to the manufacture (Promega) at 37 °C for 3 h. The digested DNA samples were loaded into a 0.8% agarose gel, containing ethidium bromide (0.3 lg/ml) and electrophoresed at 40 V.
2. Material and methods 2.1. Cell lines, viruses and insects
3. Results 2.3. Serial passage of BV 3.1. In vitro serial passage of AgMNPV-2D Three aliquots of cell suspension from each flask were collected at 72 h.p.i., and were visualized by phase contrast microscopy (Olympus CK2). The percentages of cells with many polyhedra in the nuclei (cells with MP), with few polyhedra in the nuclei (cells
The effect of serial passage of AgMNPV-2D in T. ni cells cultures was initially investigated through analysis of the percentage of cells showing occlusion bodies and the number of polyhedra pro-
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duced per cell. Ten passages of the virus in BTI-Tn5B1-4 cells were performed, and the infected cells were observed up to 72 h p.i. under a phase contrast microscope. A decrease in cell percentage with many polyhedra (MP infected cells) yield was noted as the AgMNPV-2D was serially passaged (Fig. 1). The drop was most pronounced from passage 5 to passage 6. During passaging, the percentage of cells with MP decreased while there was an increase in the percentage of cells with few polyhedra (FP infected cells). However, there was a slight increase in MP production from passage 6 to passage 8 after an initial fall. The steep slope between passage 5 and passage 6 indicates a sharp transition from an MP dominated population to a FP one. These decreases of cells with MP and increase of cells with FP is a feature of FP mutation common in prolonged viral infections in cell culture. The experiments were done by infection of 2 106 cells in 25 T flask using a fixed volume of virus (1 ml). The correlation between the virus titer obtained upon serial passage (Table 1) in those cells show a great range of the MOI. Up to the fourth passage this would represent a MOI of approximately 4. However, by the sixth passage it increases dramatically to about 1 104 viruses/cell. The polyhedra production also decreased during passage (Fig. 2). Initially, from passage 1 to passage 2, there was an increase in the number of polyhedra formed. However, after this passage the number of occlusion bodies decreased dramatically to very few polyhedra. Again the greatest decrease occurred from passages 5 to passage 6. On the other hand, BV production increased with higher passage number, reaching 1.1 1010 and 1.5 1010 PFU ml 1 at passages 5 and 7, corresponding with a gradual shift of phenotype from MP to FP phenotype. The BV titers declined at very later passage (P9) when the phenotype should have been overwhelmingly FP (Table 1).
Table 1 Budded virus production during AgMNPV-2D serial passage in cell culture. Passage number PI P3 P5 P7 P9
Virus titer(PFU/ml) 6
7.1 10 8.0 106 1.1 1010 1.5 1010 3.8 108
SD ±5.21 106 ±4.24 106 ±1.08 1010 ±8.38 109 ±1.43 108
The virus titers were determined by the 50% tissue culture infective dose (TCID50) method. The values are the averages of three determinations. The standard deviation (SD) is shown for each value.
3.2. Cell morphology alterations during virus serial passage The cell morphology of MP and FP infected cells at each passage was analyzed using phase contrast microscopy. Signs of infection,
Fig. 1. Percentage of cells that contain polyhedra during serial passage of AgMNPV2D. The percentage of cells with many polyhedra (MP) and with few polyhedra (FP) was determined after each serial passage of the virus. The values are the averages of three determinations. The vertical lines indicate the standard deviation.
Fig. 2. Polyhedra production during serial passage of AgMNPV-2D. The number of polyhedra produced per cell, within cells that produced polyhedra, was determined after each serial passage in cell culture. The values shown are the averages of three determinations. The vertical lines indicate the standard deviation.
such as larger cells and nuclear hypertrophy, presence of virogenic stroma and protrusions were observed in all cells. However, formation of occlusion bodies was not seen in all of them and was very rare in later passages. Mock infected cells were used as control (Fig. 3a.). Infected cells from passages P1, P3, P6, P8 and P10 are shown in Fig. 3 (b–f, respectively). In the initial passages (P1 and P3) some cytopathic effects as cell rounding, nuclear hypertrophy and formation of polyhedra were evident and high number of cells with MP (Fig. 3b and c) was observed. An increase in the rate of FP and reduction in MP was noted at the sixth passage (Fig. 3d). After this passage, the infected cells FP appear in lower number but appear larger. In latter passages (P8 and P10) the majority of cells are FP cells (Fig. 3e and f). The ultrastructure of infected cells was visualized by transmission electron microscopy. The cytopathic effects in infected BTI-Tn5B14 cells resulting from passages P2, P4, P6, P8 and P10 are shown in Fig. 4 (A–E, respectively). In initials passages the cells showed nuclear and cell hypertrophy with formation of many polyhedra (Fig. 4A and B) and the presence of numerous occluded virions within them (Fig. 5A). Upon serial passage the cells displayed a reduced number of polyhedra (Fig. 4C) or even had no polyhedra (Fig. 4D and E). In latter passages, as the number of polyhedra decreased the virogenic stroma became more evident with production of many virions (Fig. 4E and F).
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Fig. 3. Trichoplusia ni BTI-Tn5B1-4 cells infected with AgMNPV-2D, 72 h p.i. Phase contrast micrographs (a) Mock infected cells; passages: (b) P1, (c) P3, (d) P6, (e) P8, (f) P10. Cells were viewed with an Axiovert 135 M inverted microscope at 200 (a, d, f) and 320 (b, c, e) magnification.
The morphology of the occlusion bodies revealed that in later passages polyhedra usually have little or no virions occluded and may have aberrant morphology (Fig. 5B–D). In contrast, polyhedra with many virions (Fig. 5A) were produced at the initial passage. In addition, measurements of the relative polyhedra diameter showed that they ranged in size from 1.0 to 2.0 lm, with an average size of 1.4 ± 0.05 lm up to fifth passage and of 1.7 ± 0.05 lm at late passages. The decrease in numbers of occlusion bodies during passage in parallel to the increase of their sizes were visualized in most cells. 3.3. Restriction endonuclease analysis of virus DNA The HindIII digests of virus DNA from passages P1, P2, P4, P6 and P8 DNA were carried out to determine if gross genomic changes or rearrangements had occurred during serial passage and if this could be related to the appearance of the mutants. Comparison of the digest pattern from the first passage (P1) with the
subsequent ones showed no differences in fragment sizes (Fig. 6). No large insertions or deletions were detected. The same results were found upon digestion with BamHI (data not shown). 4. Discussion The serial passage of AgMNPV in BTI-Tn5B1-4 cells leads to accumulation of the FP phenotype. This effect has been reported for NPV groups I and II and is associated with generation of two main type of mutations, the ones termed FP mutants and the defective interfering viruses (DIs) (reviewed by Krell, 1996). In this work, the virus population clearly switched from the wild-type many polyhedra (MP) per cell phenotype to the few polyhedra (FP) per cell phenotype (Fig. 1). The FP phenotype started to accumulate on passage 6 and became predominant to the latest passage (P10). Rapid accumulation of FP mutants in cell culture has also been described for other baculovirus such as LdMNPV (Slavicek
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Fig. 5. Electron micrographs showing polyhedra and their occluded virions. (A) typical polyhedra (passage 2) with many occluded virions. (B) and (C) Polyhedra with few occluded virions (passage 8). (D) Polyhedra with aberrant morphology (passage 10).
bp 12,216 11,198 10,180 9,162 8,144 7,126 6,108
Fig. 4. Electron micrographs showing characteristics of many polyhedra phenotype and few polyhedra phenotype of AgMNPV infected cells. (A) and (B) cell with many polyhedra in the nucleus. (C) cell with few polyhedra. (D) cell with none polyhedra. (E) cell showing the virogenic stroma. (F) Detail of virogenic stroma with many virions. Panels A–F represent passage 2, 4, 6, 8, 10 and 10, respectively.
et al., 1995, 1996, 2001), HaSNPV (Lua et al., 2002) and SfMNPV (Pedrini et al., 2004). There was a tremendous 30-fold decrease in the yield of polyhedra during sequential passage and reached a phase in which occluded virus were very rare in cells (Figs. 2 and 3). This leads to a strong decrease of virulence since production of occlusion bodies is the only form of transmission of the disease in nature (Padmavathamma and Veeresh, 1991). Simultaneously, there was an increase in the yield of budded virus particles (Table 1). The stability and selective advantage of the FP mutant on serial passage are attributed to its enhanced BV production (Potter et al., 1976; Fraser and Hink, 1982; Fraser and McCarthy, 1984). FP mutants are often selected for in cell culture because they have rates of BV replication greater than those displayed by the wild-type (Harrison and Summers, 1995). The ultrastructural changes shown in infected cells serially passed with AgMNPV are typical of FP mutants. Not only the cells exhibited few polyhedra with aberrant shape, but there was minimal virion occlusion in these bodies (Figs. 4 and 5). At higher passages, instead of virus occlusion, many cells presented a well defined virogenic stroma region containing many virions, indicating intensive replication of the virus (Fig. 4E and F). Besides, polyhedra appears to grow larger during passages. These traits are the same as those exhibited by AcMNPV (Wood, 1980; Harrison and
5,090
M1
P1
P2
P4
P6
P8
M2
bp 11,500
5,000 4,600 4,500
4,072 3,054
2,800 2,600
2,036
2,100 1,900 1,700
1,636 1,100 1,000 1,018 506
800
396 344 298 220 201 154
Fig. 6. Restriction endonuclease analysis of AgMNPV-2D DNA from passages P1, P2, P4, P6 and P8. Viral genomic DNA was digested with HindIII. The fragments were separated by 0.8% agarose gel electrophoresis, and the gel was stained with ethidium bromide. Markers in base pairs: (M1) 1kb DNA ladder and (M2) DNA lambda/PstI.
Summers, 1995), TnMNPV (Potter et al., 1976), GmMNPV (Fraser and Hink, 1982), LdMNPV (Slavicek et al., 1995), HaSNPV (Lua
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et al., 2002) and SfMNPV (Pedrini et al., 2004). The polyhedra of FP mutants are reported to have much lower biological activity in in vivo assays (Potter et al., 1976; Fraser and Hink, 1982; Slavicek et al., 1995, Lua et al., 2002). Genomic restriction endonuclease digestion profiles of AgMNPV DNA from all passages were similar with no evidence of large insertions, deletions or rearrangements (Fig. 6). The fact that no significant changes were detected by REN analysis suggests that small changes such as point mutations, single nucleotide insertions or deletions, might be occurring in the AgMNPV 25kfp gene leading to FP mutants generation. However, we cannot run out the possibility that defective interfering viruses might have also been produced, at least at later passages. DIs and FP mutants induce the same cytopathic effects and similar polyhedra yield and morphology. Therefore DI particles may have contributed to some of the CPE observed. At later passages, when there was a dramatic increase in the MOI instead of only FP mutants, DIs could have been also developed which may not be evidenced at the DNA restriction profile. In addition, the up to 40-fold drop in BV production from passage 7 to passage 9 (Table 1) might have been due to DIs. In a previous study the biological constraints and bioprocess issues for in vitro production of AgMNPV was investigated (Rodas et al., 2005). For serial passage of the virus, SF9 cells (1 106 ml) were cultivated in Schott–shaker system (20 ml) and infected with a wild-type AgMNPV (not plaque purified) at MOI of 1. Analysis of the restriction profiles of viral DNA digested with HindIII and PstI enzymes showed that defective interfering particles (DIs) were formed during passaging of the virus in cell culture. However, the specific conditions used in our experiments were different. For instance, we used a different cell line (BTI-Tn5B1-4) and a plaque purified virus as inoculum (AgMNPV-2D), instead of a field isolate. It has been recently reported that while BTI-Tn5B1-4 cells are very productive for AgMNPV, the SF9 cells are much less susceptible to this virus with lower yields of BV and occlusion bodies (Castro et al., 2006). The appearance of the FP phenotype during serial passage in cell culture often correlates with the presence of DNA insertions and deletions and have been reported for AcMNPV and GmMNPV (Fraser et al., 1983; Kumar and Miller, 1987; Cary et al., 1989; Bull et al., 2003) that occur predominantly in the 25 K locus (Beames and Summers, 1989). Spontaneous insertion of transposable elements originating from host cell DNA into the viral 25kfp gene has been shown to be a common cause of the FP phenotype (Cary et al., 1989; Bull et al., 2003). In contrast to the other baculovirus FP studied, most LdMNPV FP mutants do not contain large insertions or deletions in the 25kfp gene (Slavicek et al., 1995). The majority of the FP mutants of LdMNPV contained single nucleotide insertions or small deletions (Bischoff and Slavicek, 1997). The same was described for HaSNPV (Lua et al., 2002) during passaging of this virus in cell culture. The sequences obtained from individual clones of the 25kfp gene PCR products of a late passage revealed point mutations occurring during throughout the gene. Our results seem to be similar to the ones obtained with LdMNPV and HaSNPV FP mutants. The feasibility of in vitro AgMNPV production depends on overcoming the limitations caused by virus passaging. Based in our studies the number of times that AgMNPV is passaged in cell culture should be kept to a minimum to minimize the generation of FP mutants. Therefore, preparation of large low-passage stocks is recommend. While Tn5B1-4 cells may be appropriate for production of AgMNPV producers should take care to not passage the virus more than five times. In addition, for large scale virus production a low MOI infection is required to prevent formation of DI particles.
The generation of FP mutants is a limitation for successful production of polyhedra in cell culture bioreactors. In further studies, we intend to sequence of the 25kfp gene of several FP plaque purified clones in order to characterize the most frequent mutations caused by serial passage of AgMNPV in cell culture. Also the selection of stable many polyhedra (MP) variants, competitive to FP mutants and more resistant to the passage effect, is another strategy that will be applied to the development of AgMNPV in vitro production. Acknowledgments The work is supported by grants from Embrapa and CNPq. The authors would like to thank Dr. Antonio Williams Moita, from Embrapa Hortaliças, for the statistical analysis and Dr. Bergmann Morais Ribeiro, from University of Brasilia, for electron microscope support. We also thank Dr. Peter Ward, Inglis (Embrapa Recursos Genéticos e Biotecnologia) for scientific editing of the manuscript. References Beames, B., Summers, M.D., 1988. Comparison of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra baculovirus mutants. Virology 162, 206–220. Beames, B., Summers, M.D., 1989. Location and nucleotide sequence of the 25 K protein missing from baculovirus few polyhedra (FP) mutants. Virology 168, 344–353. Bischoff, D.S., Slavicek, J.M., 1997. Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25KFP gene may be caused by DNA replication errors. J. Virol. 71, 1097–1106. Bull, J.C., Godfray, H.C., O’Reilly, D.R., 2003. A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni. Appl. Environ. Microbiol. 69, 2052–2057. Cary, L.C., Goebel, M., Corsaro, B.G., Wang, H.G., Rosen, E., Fraser, M.J., 1989. Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172, 156–169. Castro, M.E.B., Ribeiro, Z.M.A., Souza, M.L., 2006. Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: morphology, viral production and protein synthesis. Biol. Control 36, 299–304. Chakraborty, S., Reid, S., 1999. Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures. J. Gen. Virol. 73, 303– 308. Fraser, M.J., Hink, W.F., 1982. The isolation and characterization of the MP and FP plaque variants of Galleria mellonella nuclear polyhedrosis virus. Virology 117, 366–378. Fraser, M.J., McCarthy, W.J., 1984. The detection of FP plaque variants of Heliothis zea. J. Invertebr. Pathol. 43, 427–429. 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. J. Virol. 47, 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. Granados, R.R., Li, G., Derksen, A.C.G., McKenna, K.A., 1994. A new cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J. Invertebr. Pathol. 64, 260–266. Harrison, R.L., Summers, M.D., 1995. 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. J. Gen. Virol. 76, 1451–1459. Herniou, E.A., Olszewski, J.A., Cory, J.S., O’Reilly, D.R., 2003. The genome sequence and evolution of baculoviruses. Annu. Rev. Entomol. 48, 211–234. Hink, W.F., Vail, P.V., 1973. A plaque assay for titration of alfalfa lopper nuclear polyhedrosis virus in a cabbage lopper (TN 368) cell line. J. Invertebr. Pathol. 22, 168–174. Johnson, D.W., Maruniak, J.E., 1989. Physical map of Anticarsia gemmatalis nuclear polyhedrosis virus (AgMNPV-2) DNA. J. Gen. Virol. 70, 1877–1883. Kool, M., Voncken, J.W., van Lier, F.L., Tramper, J., Vlak, J.M., 1991. Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties. Virology 183, 739–746. Krell, P.J., 1996. Passage effect of virus infection in insect cells. Cytotechnology 20, 125–137. Kumar, S., Miller, L.K., 1987. Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture. Virus Res. 7, 335–349. Lee, H.Y., Krell, P.J., 1992. Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus. J. Virol. 66, 4339–4347.
S. H. M. S. d. Rezende et al. / Journal of Invertebrate Pathology 100 (2009) 153–159 Lua, L.H.L., Pedrini, M.R.S., Reid, S., Robertson, A., Tribe, D.E., 2002. Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. J. Gen. Virol. 83, 945–955. Moscardi, F., 1999. Assessment of the application of baculoviruses for control of lepidoptera. Ann. Rev. Entomol. 44, 257–289. Moscardi, F., Souza, M.L., 2002. Baculovírus para o controle de pragas: Panacéia ou realidade? Biotecnol. Ciênc. Des. 24, 22–29. Oliveira, J.V.C., Wolff, J.L.C., Garcia-Maruniak, A., Ribeiro, B.M., Castro, M.E.B., Souza, M.L., Moscardi, F., Maruniak, J.E., Zanotto, P.M.A., 2006. Genome of the most widely used viral biopesticide: Anticarsia gemmatalis multiple nucleopolyhedrovirus. J. Gen. Virol. 87, 3233–3250. O’Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus Expression Vectors: A Laboratory Manual. Oxford Univ. Press, New York. Padmavathamma, K., Veeresh, G.K., 1991. Effect of larval age and dosage of nuclear polyhedrosis virus on the susceptibility of diamondback moth, Plutella xylostella. Entomol. Exp. Appl. 60, 39–42. Pedrini, M.R.S., Wolff, J.L.C., Reid, S., 2004. Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures. Ann. Appl. Biol. 145, 107–112. Pedrini, M.R.S., Nielsen, L.K., Reid, S., Chan, L.C.L., 2005. 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. In vitro Cell. Dev. Biol. Animal 41, 289–297. Pijlman, G.P., Born, E.V.D., Martens, D.E., Vlak, J.M., 2001. Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283, 132–138. Potter, K.N., Faulkner, P., MacKinnon, E.A., 1976. Strain selection during serial passage of Trichoplusia ni nuclear polyhedrosis virus. J. Virol. 18, 1040–1050. Reed, L., Muench, H., 1938. A simple method for estimating fifty percent endpoints. Am. J. Hygenf. 27, 493–497. Rodas, V.M., Marques, F.H., Honda, M.T., Soares, D.M., Jorge, S.A.C., Antoniazzi, M.M., Medugno, C., Castro, M.E.B., Ribeiro, B.M., Souza, M.L., Tonso, A., Pereira, C.A.,
159
2005. Cell culture derived AgMNPV bioinsecticide: biological constraints and bioprocess issues. Cytotechnology 48, 27–39. Russell, R.L.Q., Rohrmann, G.F., 1993. A 25-kDa protein is associated with the envelopes of occluded baculovirus virions. Virology 195, 532–540. Slavicek, J.M., Podgewaite, J., Lanner-Herrera, C., 1992. Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide Gypchek. J. Invertebr. Pathol. 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. Biol. Control 5, 251–261. Slavicek, J.M., Mercer, M.J., Kelly, M.E., Hayes-Plazolles, N., 1996. Isolation of a baculovirus variant that exhibits enhanced polyhedra production stability during serial passage in cell culture. J. Invertebr. Pathol. 67, 153–160. Slavicek, J.M., Hayes-Plazolles, N., Kelly, M.E., 2001. Identification of a Lymantria dispar nucleopolyhedrovirus isolate that does not accumulate few polyhedra mutants during extended serial passage in cell culture. Biol. Control 22, 159– 168. Theilmann, D.A., Blissard, G.W., Bonning, B., Jehle, J., O´Reilly, D.R., Rohrmann, G.F., Thiem, S., Vlak, J.M., 2005. Baculoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Eighth Report of the International Committee on Taxonomy of Viruses. Eselvier Academic Press, Amsterdam, pp. 177–185. Wickham, T.J., Davis, T., Granados, R.R., Hammer, D.A., Shuler, M.L., Wood, H.A., 1991. Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection. Biotechnol. Lett. 13, 483–488. Wood, H.A., 1980. Isolation and replication of an occlusion body-deficient mutant of the Autographa californica nuclear polyhedrosis virus. Virology 105, 338–344. Wu, D., Deng, F., Sun, X., Wang, H., Yuan, L., Vlak, J.M., Hu, Z., 2005. Functional analysis of FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. J. Gen. Virol. 86, 2439–2444. Zanotto, P.M., Kessing, B.D., Maruniak, J.E., 1993. Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J. Invertebr. Pathol. 62, 147–164.