Applied and Environmental Microbiology, April 2003, p . 2052-2057, Vol . 69, No . 4

A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

James C . Bull,^1* H . C . J . Godfray,² and David R . O'Reilly^1,1

Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ,¹ NERC Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, United Kingdom²

Received 28 May 2002/ Accepted 23 September 2002

Mutations affecting the production of OBs make good candidates for viral traits where such rescue is possible . The process of co-occlusion, in which a wild-type genotype and an occlusion-negative (occ^- genotype are incorporated into the same OB (11), has been investigated as a potential strategy for application of genetically modified viral genomes for insecticidal purposes (11, 18, 29) . In such a case, there is inevitable loss of the occ^- genotype during serial rounds of insect infection . This is because cells that are infected solely by occ^- genomes do not produce OBs, and so a fraction of the occ^- population is lost . However, it has been suggested that an advantageous replication rate of BVs might offset this disadvantage and allow a stable equilibrium between wild-type and parasitic mutant genotypes (10) . A well-described class of mutants that have characteristics suitable for testing this hypothesis is the few-polyhedra (FP) mutant form of NPVs . FP mutants are only known from serial passage of NPVs in cell culture (14) . They classically yield very small quantities of OBs (28) and poorly occlude virions (12) . In this respect, they are effectively occ^- . However, these aspects of the virus phenotype are of little importance in cell culture, and FP mutants are often selected for because they have rates of BV replication greater than those displayed by the wild type (12) .

FP mutants have not been obtained from field collections of NPVs, but this may simply be because they are eliminated when clonal lines of viruses isolated from field collections are purified prior to characterization . A defective strain was isolated from a field isolate of Spodoptera exigua NPV and was shown to be parasitic on the viral population (21) . In order to ascertain the likelihood that strains such as this strain exist as parasitic mutants in the field, it is important to establish a quantitative framework to investigate the conditions under which they may persist . A model of NPV population genetics was developed, in which the distribution of the multiplicity of infection (MOI) of cells by viral genomes is an important predictor of viral strain coexistence (10) . In laboratory-based experiments workers found a mean MOI of more than four genomes per cell, with a Poisson distribution (4) . In this case, only a relatively modest BV replication advantage for the mutant strain would allow it to persist at a detectable level in stable equilibrium with the wild type . Here we tested this prediction with an FP mutant isolated from cell culture .

We isolated an FP mutant from routinely passaged Autographa californica MNPV (AcMNPV) and determined its phenotype . This new virus displayed classic FP characteristics, with infected cells typically producing fewer than 10 OBs each, compared to as many as 100 OBs produced by cells infected with the wild type (9) . Electron microscopy revealed that the OBs only very rarely showed signs of occluding virions . As expected from this OB morphology, the mutant proved to be largely noninfectious to insects per os . However, the virus displayed a rate of BV replication that was 1.6 times that of the wild type . We tested the prediction that an FP mutant might be able to persist in a stable equilibrium along with a wild-type strain by coinfecting fifth-instar Trichoplusia ni larvae with a wild-type virus and the FP mutant strain during serial rounds of infection . Insects were coinfected by the two strains, which were introduced at a 1:1 ratio, and were passaged through six rounds of serial infection . In contrast to the findings obtained for co-occlusion with an artificial occ^- strain showing no replicative advantage (4), the relative frequency remained stable at this proportion .

Electron microscopy.
TN368 cells were infected with either L1 or fp-1 at a MOI of 10 PFU per cell . At 48 h postinfection, infected cells were harvested, washed in phosphate-buffered saline, and resuspended in 1.5 ml of phosphate-buffered saline . The cells were then fixed in 1 ml of 4% paraformaldehyde-1% gluteraldehyde-0.1 M phosphate buffer . Samples were sequentially dehydrated in 30, 50, 70, and 80% ethanol . Following dehydration, the cells were sequentially infiltrated with LR white resin (London Resin Co., Reading, United Kingdom) by adding 200-µl portions of 50, 75, and 100% resin in ethanol . The cells were resuspended in 200 µl of fresh 100% resin and transferred to size 4 gelatin capsules (Agar Scientific, Stansted, United Kingdom) . These capsules were incubated at 50°C for 18 h to set .

Sections were cut from the resin blocks with a Reichert-Jung Ultracut ultramicrotome set to a thickness of 70 nm . These sections were then stained with 2% uranyl acetate and lead citrate precipitate and left to dry (23) . Sections were visualized and photographed with a Philips 400 transmission electron microscope .

Insects and bioassays.
T . ni eggs were obtained from J . Cory (NERC Centre for Ecology and Hydrology, Oxford, United Kingdom) . Larvae were reared on an artificial diet at 27°C (2) . On the first day of the 5th-instar stage, larvae were infected with virus either by injection of BVs or per os with OBs . For injection, 5-µl aliquots were injected subcutaneously with a Hamilton syringe . When hemolymph was collected, it was collected by removing a hind proleg and bleeding onto Parafilm . Insects were infected per os by applying a suspension of OBs to cubes of artificial diet that were approximately 2 mm in diameter . The insects were starved for 6 h before the infected diet cubes were presented . When the infected diet had been consumed, the larvae were refed with artificial diet as necessary until they died . In all insect infection experiments, a number of uninfected insects were retained until pupation as controls . Preliminary bioassays with purified OBs were carried out to assess the dose-response relationship between AcMNPV and fifth-instar T . ni larvae under our experimental conditions (data not shown) .

Construction of revertant virus.
A revertant virus strain that was designated AcMNPV.rev (referred to as rev below) was generated by recombination between fp-1 and a PCR-amplified DNA sequence that included the wild-type fp25 gene . This sequence extended from position 47766 to position 49987 and was generated by using primers fprevert (5'-ggaaTTCAGTGTCATAATCCGTG-3') and fptrever (5'-ggaATTCCAGTCGGTAGATGAC-3') (the bases in lowercase letters are not part of the genomic sequence and were added to the primers in order to create EcoRI restriction enzyme recognition sites [underlined]) . The procedure was designed so that a sequence could be incorporated into a plasmid if transfection of the PCR product alone proved to be ineffective . This was not necessary . Following cotransfection of IPLB-Sf21-AE cells with viral and PCR-amplified DNA, the progeny virus was screened by a plaque assay for wild-type recombinants . Phenotypically correct clones were verified by restriction enzyme analysis .

Infectivity bioassay.
A bioassay was carried out in order to compare the infectivities of the L1, fp-1, and rev strains of AcMNPV . Serial dilutions containing 10², 10³, 10⁴, 10⁵, and 10⁶ OBs in 1 ml of distilled water were prepared for each of the three viruses, and 50-µl aliquots were spread onto the surfaces of individual wells filled with artificial diet, so that 10 replicates at each dilution were created for each virus . In addition, 50-µl aliquots of distilled water were spread onto the surfaces of 10 wells to act as negative controls . Individual T . ni larvae were placed in separate wells on the second day after hatching . The insects were incubated at 27°C for 7 days . Following incubation, the numbers of dead insects were recorded for each of the virus dilutions, and the percentages of mortality were calculated .

Viral replication rates.
In order to assess the relative rates of replication of the wild-type and FP mutant viruses, insects were infected by a single viral strain, and the titer of the viral progeny was measured over the course of the infection . For each viral strain, 12 insects were infected by injection of 10⁴ PFU (total dose), and hemolymph was collected from sets of three insects at 24, 48, 72, and 96 h postinfection (at 96 h postinfection all insects were dead) . The titer of each viral strain in the hemolymph was measured by a plaque assay .

Southern blot analysis.
Viral DNA from OBs formed in a cell culture was purified by the mini-prep method as described previously (22) by using sequential phenol, phenol-chloroform, and chloroform-indoleacetic acid extraction and ethanol precipitation . Viral DNA from OBs formed in insects was also purified as described previously (22), except that dialysis in 0.1x Tris-EDTA for 2 h at 4°C was included before ethanol precipitation .

Viral DNA was digested for 4 h with HindIII, and the fragments were separated by electrophoresis . The genotypes could be distinguished by analysis of the resulting patterns . A 518-bp DNA sequence corresponding to part of the flanking region downstream of the fp25 gene was generated by PCR with the fpprobe primer (5'-GTGCGAGACGCCTTGG-3') and the fpeborp primer (5'-CGTCGAGACGTTTACAAAG-3') and used as a probe . It was labeled with [-³²P]dCTP (3,000 Ci/mmol) by using a High Prime DNA labeling kit (Roche Diagnostics Ltd., Lewes, United Kingdom), and 0.8% agarose gels were transferred to Hybond N+ membranes (Amersham Pharmacia Biotech Inc., Herts, United Kingdom) by capillary action as described previously (24) . Hybridization was performed overnight at 65°C in fresh 5x SSC-0.5% SDS-6x Denhardt's solution containing 100 µg of denatured, fragmented salmon sperm DNA per ml plus radiolabeled probe (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) . The blots were washed once in 1x SSC-0.5% SDS for 20 min at 65°C and then twice in 0.5x SSC-0.5% SDS for 20 min at 65°C . The blots were exposed for 1 h to BAS-MP plates, which were then scanned with a BAS-1500 phosphorimager (Fujifilm) . The probe extended from position 47511 to position 48029 (1) of the AcMNPV genome, within the HindIII I fragment, and hybridized to a 5.0-kb fragment in the wild-type digest and to a 5.3-kb fragment in the FP mutant digest . The relative amounts of wild-type and FP mutant genomes in a preparation were therefore determined by comparing the radiation intensities of the bands by using MacBas, version 2.5, software . In trial experiments, different amounts of wild-type DNA, quantified with a Beckman DU 640 spectrophotometer, were digested and analyzed as described above to confirm that there was a linear response (data not shown) .

Serial rounds of insect infection.
In order to track the fate of viral genomes in coinfection during serial rounds of insect infection, 10 insects were infected by injection of the two genotypes at a 1:1 ratio by using a total dose of 10⁴ PFU in 5 µl . OBs were harvested after death, purified to disrupt any nonoccluded virus, and pooled . This preparation was used as the starting inoculum for subsequent rounds of infection, in which the insects were infected with OBs per os . This was done by using a high dose (5,000 OBs per insect) for five additional rounds of serial infection for 10 independent lines of insects . For each round of infection, OBs were taken from a single cadaver for each insect line and used to infect three insects for the next round of infection to ensure continuation of that line . Viral DNA was collected from OBs obtained from every round of infection (from the single insect selected from each line for the next round of infection), and the ratio of the genotypes in each sample was determined from the intensity of hybridization following Southern blotting as described above .

Analysis.
We assumed that multiple genomes got into the host, so that the frequency of the FP mutant (fp-1) at the beginning of any particular round of infection was p . The fp-1 BVs had a replication advantage (W), which led to an increase in the frequency (p*), as given by the equation

 
As expected from the OB morphology, the bioassay revealed that fp-1 had greatly reduced infectivity for T . ni larvae when it was administered per os in the form of OBs (Fig . 2) . The revertant strain, rev, displayed wild-type infectivity .

 
Relative replication rates of L1, fp-1, and rev.
A key parameter in the model that was predicted to affect the persistence of genotypes was the relative replication rates of the viruses . We expected to see a difference in the replication rates of the virus genotypes, and to quantify this, the replication rates of the viruses in insects were compared . T . ni larvae were infected by injection, and hemolymph was collected from groups of three insects at various times postinfection and used for a plaque assay . Figure 3 shows that there was not a significant difference in the titers of L1 and rev at any time after infection . However, Fig . 3 shows that fp-1 had a replication advantage of 1.6 times relative to the replication of L1 .

 
Persistence of fp-1 during serial passage.
Initially, 10 insects were infected with equal amounts of the L1 and fp-1 viruses by injection, and OBs were recovered and pooled . The OB dose (more than 10 times the 100% lethal dose) (dose-response data not shown) was chosen so that a large number of virions could be assumed to have crossed the insect gut wall and to have initiated infection . This should have ensured that the mean relative frequencies of the genotypes in the OB inoculum were the same as those which initiated host tissue infection . The mean ratio of the genotypes in these insects was found to be 45% L1 to 55% fp-1 . In order to track the fate of the viral genotypes during serial rounds of infection, 10 insects were infected per os by using this OB preparation . A further five serial rounds of infection were carried out with 10 independent lines of insects, and the viral genotype ratios were determined by Southern blotting for all insect lines after each round of infection . Figure 4 shows that the proportion of fp-1 remained stable with passage . The percentage of the wild type, which was consistent for all five subsequent rounds of coinfection, was 48.4% ± 1.62% (mean ± standard error; n = 10) . We investigated what mean MOI of cells within the insect could best account for this persistence of fp-1, assuming a Poisson distribution and the measured replication advantage (by using the model described in Materials and Methods) . We found that a mean of 4.27 genomes per cell provided the best fit to the data . This is in line with the distribution of MOI reported previously (4) and the predictions of the model (10) .

 
Baculovirus biology can be significantly influenced by coinfection of cells with multiple viral genomes, which facilitates phenomena such as complementation or recombination between genomes, interference by defective genomes, and viral evolution . Until recently, virtually nothing was known about the frequency of coinfection at the cellular level in infected insects . We have previously reported a surprisingly high frequency of multiple cell infection based on the persistence of an occ^- recombinant strain of AcMNPV when it was co-occluded with wild-type virus during serial rounds of insect infection (4) . In addition, a strain of S . exigua NPV, isolated from the field, was shown to be parasitic on the predominant strain (21) and persisted at a stable level for four rounds of serial infection in test populations of insects as part of the natural virus population (20) . However, no mechanism for this persistence was suggested .

Here, we demonstrated that an occ^- strain, which has a replication advantage over wild-type virus, could persist as a stable polymorphism with the wild type, in which the relative frequencies of the genotypes were dependent on the replication advantage of the occ^- strain . This is an interesting result in itself as it is the first reported case of an FP mutant existing within a stable polymorphism over serial rounds of insect infection . In addition, a Poisson distribution of MOI with a mean of 4.3 virus genomes per cell provides a very good fit to the data, which is in agreement with the results reported for trials performed with the artificial, occ^- recombinant (4) .

In these trials, the FP mutant could described as parasitic on the wild-type strain, and here at least, the situation appeared to be stable . While the FP phenotype is a phenomenon of cell culture and has not been reported in field collections of NPVs, the results support the idea that occlusion-deficient mutants might persist in the field . Natural populations of NPVs are often known to comprise several closely related strains (3, 7, 16, 25), but there is little information about the temporal dynamics or about the relative fitness of or interactions between the coinfecting strains .

Nearly all eukaryotes, prokaryotes, and viruses go through a stage in their life cycles in which only a single copy of the genome is present . Evolutionary theorists have argued that this is a fundamental adaptation of living organisms to prevent damage due to parasitic genomes (17) . Previous work has shown that cells producing OBs are infected by multiple viral genomes, and thus NPVs appear to be an exception to this generalization . The work reported here shows that a common type of genomic parasite that appears in artificial NPV cell cultures is also maintained during serial propagation in insects . The challenge now is to evaluate whether this type of genomic parasite also occurs in insect-NPV interactions in the field .

We thank Ian Morris and Renée Lapointe for help with preparation of electron micrographs .

Present address: Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom .

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