In Vivo Recombination between Two Strains of the Genus ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1997, p. 3025–3031 0099-2240/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 63, No. 8

In Vivo Recombination between Two Strains of the Genus Nucleopolyhedrovirus in Its Natural Host, Spodoptera exigua ˜ OZ,1 JUST M. VLAK,2 DELIA MUN

AND

PRIMITIVO CABALLERO1*

Laboratorio de Entomologıá Agrıćola y Patologıá de Insectos, Departamento de Produccio ń Agraria, Campus de Arrosadıá s/n, Universidad Pu ´blica de Navarra, 31006 Pamplona, Spain,1 and Department of Virology, Wageningen Agricultural University, 6709 PD Wageningen, The Netherlands2 Received 23 December 1996/Accepted 20 May 1997

The DNA restriction map for the enzymes BamHI, BglII, PstI, and XbaI of SeMNPV-US (Se-US), the best-studied Nucleopolyhedrovirus strain isolated from Spodoptera exigua in California, was used as a reference to construct that of SeMNPV-SP2 (Se-SP2), a closely related Spanish strain of the same virus. After coinfection of S. exigua with both the Se-US and Se-SP2 strains per os, a recombinant virus (SeMNPV-SUR1 [Se-SUR1]) was detected after one passage which quickly replaced the parental viruses. A physical map of Se-SUR1 DNA was constructed for BamHI, BglII, PstI, and XbaI and compared to that of the parental viruses, Se-US and Se-SP2. Se-SUR1 is the result of at least four crossover events between Se-US and Se-SP2 and not of selection of a minor variant in one of the parental viruses. Bioassays of the recombinant and parental strains against L2 beet armyworm larvae demonstrated that their 50% lethal dose values were not significantly different. The differences in median lethal time values are too small to explain the replacement of the parental viruses by the recombinant virus upon successive passage in vivo, although it cannot be ruled out as an explanation for the selective advantage of the recombinant strain, Se-SUR1. The consequences of the release of nonindigenous or recombinant baculovirus strains in agro-ecosystems are discussed. land), the PstI M fragment from each isolate served as a restriction fragment length polymorphic marker for their identification (6). Additionally, the biological activities of some of the various SeMNPV strains differed significantly from each other. Hara et al. (15) reported four different 50% lethal dose (LD50) values for six strains studied. Likewise, Caballero et al. (6) showed three distinct LD50s for the four strains they analyzed. Additional information to differentiate strains can be obtained by virion protein profiles and production of occlusion bodies (OBs) in cell culture (15). The SeMNPV is a more effective control agent for S. exigua than any other Nucleopolyhedrovirus (26). Recently, a Nucleopolyhedrovirus which originated from Florida has been developed commercially as a specific S. exigua larva bioinsecticide for use on field and greenhouse crops (20, 27). An important feature of such a virus is its capacity to replicate in and spread through host populations. Consequently, applications of an exotic SeMNPV strain to reduce damage by S. exigua might replace or alter other indigenous SeMNPV populations that are present at enzootic or epizootic levels. To better evaluate and ascertain this possibility, it is important to monitor the viral progeny in larvae coinfected by two different and distinguishable virus strains. In the present study, our aim was to test the potential impact which the introduction of wild-type or genetically altered baculoviruses might have on populations of natural insect baculoviruses of the genus Nucleopolyhedrovirus, specifically of SeMNPV.

Baculoviruses of the genera Nucleopolyhedrovirus and Granulovirus have been isolated worldwide from a large number of insect species (24). Under natural conditions, baculoviruses have been demonstrated on numerous occasions to regulate insect populations, including those of major pest species (29). Baculoviruses are considered useful and safe candidates for biological control (16, 25), and as a consequence, some have been developed by industry, registered in several countries, and marketed for use in agriculture and forestry. These products and other noncommercial preparations of baculoviruses have been used successfully as bioinsecticides to control many species of lepidopteran pests (18, 25), with minimal impact on nontarget invertebrate natural enemies (3). The beet armyworm, Spodoptera exigua, is a major caterpillar pest of agricultural crops in tropical and subtropical regions (4) and shows resistance (2, 7) or high tolerance (30) to most of the available chemical insecticides. A Nucleopolyhedrovirus strain isolated from S. exigua (SeMNPV) has been reported as a promising control agent since it is effective against its natural host on various field and greenhouse crops by inundative spraying of the virus (13, 27). Other Nucleopolyhedrovirus isolates from S. exigua have been collected in Egypt and The Netherlands (32), California (14), Spain (5), and Japan and Thailand (15, 21), where this pest usually needs to be controlled to avoid crop losses. DNA restriction endonuclease analysis (REN) of different SeMNPV isolates confirmed that although they were closely related, most were distinct strains. The strains can be distinguished unequivocally from each other by one or more DNA restriction enzyme fragments. For example, for SeMNPV strains from three geographically very separate regions (from the United States, Spain, and Thai-

MATERIALS AND METHODS Viruses. Strain SeMNPV-US (Se-US) was from M. D. Summers (Texas A&M University, College Station). SeMNPV-SP2 (Se-SP2) was isolated during the course of a viral epizootic in vegetable greenhouses in southern Spain (6) and was maintained in this laboratory.

* Corresponding author. Phone: 34 48 169129. Fax: 34 48 169169. E-mail: [email protected]. 3025

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APPL. ENVIRON. MICROBIOL.

FIG. 1. (a) Restriction enzyme profiles of Se-US and Se-SP2 (left and right lanes, respectively) DNA with BamHI, BglII, PstI, and XbaI as indicated. All DNA bands are marked with their corresponding letter, and for those unique to each strain, the length in kilobases is shown in brackets. (b) Physical maps of Se-SP2 and Se-US DNA for BamHI, BglII, PstI, and XbaI and corresponding scale (bottom row) in kilobases and map units. (c) Physical map of Se-SUR1 DNA for BamHI, BglII, PstI, and XbaI. The position and origin of diagnostic fragments are shown for Se-SP2 (open bars), Se-US (hatched bars), and Se-SUR1 (closed bars). (d) Simplified organizational map of Se-SUR1 DNA. Areas where the crossovers could have occurred (closed bars), Se-SP2 marker fragments (open bars), and Se-US markers (hatched bars) are indicated.

Insect rearing. S. exigua cultures were reared on a semisynthetic diet (18% corn flour, 3.4% brewer’s yeast, 3.2% wheat germ, 1.5% casein, 0.45% ascorbic acid, 0.11% nipagine, 0.05% formaldehyde, 0.13% benzoic acid, and 1.5% agar) at 27 6 2°C, in 70 to 80% humidity, and at a 16-h-light–8-h-dark photoperiod. Propagation of viruses and mixed infections. A fresh stock of OBs of each SeMNPV isolate was obtained by feeding early-fourth-instar (L4) S. exigua larvae with artificial diet contaminated with the appropriate virus. The infected larvae were reared on formaldehyde-free diet and collected after death (ca. 4 to 6 days after infection). To produce enough inocula of both parental viruses for the different experiments, OBs were recovered from a pool of larvae and purified, first by filtration through cheesecloth to remove the larval debris and then through a 30% (wt/vol) sucrose layer by centrifugation at 6,800 3 g for 10 min in a tabletop centrifuge. OB pellets were washed twice with water to remove the sucrose. The polyhedra were stored at 220°C until used. In all cases, larvae were infected with SeMNPV OBs at a dose of 106 OBs per larva. For coinfection of newly molted L4 S. exigua larvae, small diet plugs were contaminated with a 1:1 mixture of an OB suspension of Se-US and Se-SP2 that had been reported to have similar insecticidal activities (6). As controls, an additional cohort of larvae was infected at the same time with Se-US and another was infected with Se-SP2. After approximately 24 h, when the larvae had consumed all the food, they were fed with uncontaminated diet until death. All larvae which died of polyhedrosis were collected individually and kept at 220°C for further analysis. OBs were recovered from these larvae and purified as indicated above. For successive infections, OBs obtained from the previous virus passage were fed to healthy L4 S. exigua larvae. Isolation of viral DNA, REN analysis, and gel electrophoresis. Viral DNA was isolated from approximately 109 OBs in 300 ml of water. OB suspensions were treated with a one-third volume of 33 DAS (0.3 M Na2CO3, 0.5 M NaCl, 0.03 M EDTA [pH 10.5]) at 37°C for 5 min to dissolve the polyhedrin matrix. Undissolved OBs and other heavy particulate material were pelleted by centrifugation at 6,800 3 g for 8 min. The virion-containing supernatant was transferred to sterile microcentrifuge tubes and incubated with proteinase K (200 mg/ml) at

45 to 50°C for 2.5 h and then with 1% sodium dodecyl sulfate (SDS) for an additional 0.5 h. Viral DNA was extracted once with an equal volume of TE (10 mM Tris, 1 mM EDTA [pH 8.0]) buffer-saturated phenol and twice with equal volumes of TE buffer-saturated phenol-chloroform-isoamyl alcohol (25:24:1). The DNA suspension was dialyzed against three to four changes of 0.013 TE at 4°C for 48 h. The DNA yields ranged between 60 and 90 mg. Between 1 and 2 mg of viral DNA was incubated with 10 U of restriction enzyme (Amersham), as described in the supplier’s instructions, at 37°C for 4 h. Reactions were stopped by the addition of one-sixth volume of 63 loading buffer (0.25% bromophenol blue, 40% sucrose) and then loaded onto 0.8% agarose gels in TAE (40 mM Tris-acetate, 1 mM EDTA [pH 8.0]) containing 0.25 mg of ethidium bromide per ml. Southern blot hybridization. For Southern blotting, digested DNA was electrophoresed in 0.8% agarose gels, and following electrophoresis, the gel was incubated in 0.25 M HCl for 15 min and then rinsed. DNA was denatured with 0.5 M NaOH for another 15 min and neutralized with 203 SSC (3 M NaCl, 0.3 M sodium citrate [pH 7.0]) for 30 min. Southern blotting was done overnight on HyBond membranes with 203 SSC as the elution solution. After blotting, DNA was cross-linked to the membrane by a 1-min exposure to UV light. Polymorphic fragments from Se-US and Se-SP2 were recovered from 0.8% agarose gels and purified by use of the Glass-MAX kit (Life Technologies). Approximately 50 ng of DNA was radiolabelled with [a-32P]dATP by random priming with a kit (Boehringer-Mannheim). Blots were prehybridized in Church buffer (7% SDS, 10 mg of bovine serum albumin per ml, 0.25 M Na3PO4 [pH 7.2], 1 mM EDTA) for 30 min at 65°C. Denatured probe was then added, and hybridization proceeded overnight at the same temperature. Blots were washed with 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% SDS for 5 min at room temperature with 23 SSC–0.1% SDS for 5 min at room temperature, and with 0.13 SSC–0.5% SDS for 1 h at 65°C. Autoradiography was done with Hyperfilm-ECL (Amersham) and an intensifying screen at 220°C for 2 to 24 h.

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FIG. 1—Continued.

Bioassays. The LD50s and median lethal times (LT50s) of the parental strains Se-US and Se-SP2 and of the recombinant strain SeMNPV-SUR1 (Se-SUR1) were determined in a bioassay after the droplet-feeding method of Hughes and Wood (19). Second-instar S. exigua larvae were starved for 16 to 20 h at 25°C. These larvae were then allowed to drink from an aqueous suspension containing 10% (wt/vol) sucrose, 0.001% (wt/vol) Fluorella blue, and OBs at concentrations of 8.18 3 104, 2.72 3 104, 9.09 3 103, and 3.03 3 103/ml. Larvae under such conditions are calculated to ingest a mean volume of 0.33 ml (7), so that the mean ingested doses of virus were calculated to be 27, 9, 3, and 1 OBs/larva, respectively. This concentration range was found to kill between 5 and 95% of the test

larvae in preliminary bioassays. Twenty-microliter droplets of a given concentration of OBs were applied to the bottom of a petri dish. The first larvae that drank from the solution within 10 min were transferred to individual wells of a 25-well tissue culture plate with a semisynthetic diet plug. Twenty-five larvae were used per replica, and the whole bioassay was repeated twice. A cohort of 75 larvae was allowed to drink from an OB-free suspension and served as a control. Larvae were reared at 25°C, and mortality was recorded every 12 h until they had either died or pupated. The dose mortality data were processed and analyzed with the computer program POLO-PC (Le Ora Software Inc., Berkeley, Calif.). This program is

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based on the probit analysis method described by Finney (12). LT50s were calculated by use of the mathematical equation described by Biever and Hostetter (1).

RESULTS REN and restriction fragment length polymorphism analysis of Se-US and Se-SP2. DNA profiles of both parental viral strains were obtained with BamHI, BglII, HindIII, PstI, SpeI, SstI, and XbaI and compared. Gel patterns resulting from HindIII, SpeI, and SstI were identical (data not shown) and resembled those reported by Heldens et al. (17). However, several polymorphic fragments which distinguished both viruses were obtained with BamHI, BglII, PstI, and XbaI (Fig. 1a). REN analysis of the Se-SP2 viral preparation indicated many bands in common with those of the Se-US strain. In addition to mostly equimolar DNA fragments, some of the Se-SP2 fragments were present at a submolar level, suggesting heterogeneity within the Se-SP2 field isolate. For example, Se-SP2 bands for BamHI fragments D and E, BglII fragment K, PstI fragment M, and XbaI fragments M and Q were submolar. Other submolar bands were also observed but were not used as reference markers. These are indicated by a subscript and included BamHI fragment B1, BglII fragment J1, PstI fragment L1, and XbaI fragment M1 but were not used in the construction of the physical map. A physical map of Se-SP2 was constructed to aid in identification and mapping of those polymorphic fragments that could be useful as markers to help differentiate different genotypes and, in particular, to aid in the analysis of potential recombinants between Se-US and Se-SP2. Physical map of the Se-SP2 genome. The Se-SP2 restriction enzyme map for BamHI, BglII, PstI, and XbaI was constructed by use of the physical map of Se-US as a reference (17). For BglII, reciprocal hybridization between the Se-SP2 and Se-US strains confirmed the identity of equal-sized fragments between the two strains, and these were mapped to collinear positions on the Se-SP2 map. For others, DNA fragments which were of the same size for both strains were presumed to be identical and were also mapped to collinear positions. Polymorphic fragments of Se-SP2 were used as probes against Se-US DNA (digested with all seven enzymes used in the Se-US physical map) and Se-SP2 DNA cleaved with BamHI, BglII, PstI, and XbaI to map their relative positions. The SeSP2 polymorphic BamHI F and H fragments both hybridized to Se-US BamHI fragment F. The Se-SP2 polymorphic XbaI fragment Q hybridized to Se-US XbaI fragment Q, while both Se-SP2 XbaI fragments R and O hybridized to Se-US XbaI fragment K. Se-SP2 PstI fragment H hybridized to adjacent Se-US PstI fragments K and N. Se-SP2 BglII fragments L and K hybridized to Se-US BglII fragments J and H, respectively; Se-SP2 BglII fragments C, E, F, and P hybridized to Se-US BglII fragment A. Se-SP2 BglII fragment N hybridized to Se-US BglII fragment L, and both Se-SP2 BglII fragments M and O hybridized to Se-US BglII fragment K. From the results of these and other hybridizations and by use of the estimated sizes of the Se-SP2 restriction enzyme DNA fragments, we constructed a physical map of the Se-SP2 strain (Fig. 1b). The Se-SP2 map was oriented with the entire XbaI S fragment to the left since this fragment contains the polyhedrin gene (31). Digestion with BglII resulted in the greatest difference among restriction enzyme profiles of DNA from the two SeMNPV viruses, with the Se-SP2 strain DNA having more BglII sites than the Se-US strain did. In the Se-SP2 region equivalent to the Se-US 36-kb BglII A fragment, there are an additional three BglII sites resulting in Se-SP2 BglII subfragments C, E, F, and P. Similarly, there is an additional BglII site

FIG. 2. BglII profiles of viral DNA from the first coinfection of larvae with Se-US and Se-SP2. Lanes: 1, Se-US; 2 to 10, viral DNA from individual coinfected larvae; 11, Se-SP2. A novel 10.6-kb BglII submolar fragment not present for either Se-SP2 or Se-US is indicated by an arrow in lane 5.

in the Se-SP2 region corresponding to Se-US BglII fragment K in which this 3.1-kb fragment is resolved into Se-SP2 BglII fragments M and O. The Se-SP2 BglII K fragment is about 0.3 kb shorter than the corresponding Se-US BglII H fragment. Only two differences were detected in the PstI map. The first coincides with a missing PstI site for the Se-SP2 isolate, reflected by the presence of a longer PstI H fragment and corresponding to a fusion of Se-US PstI fragments K and N. The second concerns the Se-SP2 PstI M fragment, which is 0.3 kb shorter than that of Se-US PstI fragment M. Both Se-SP2 PstI fragment M and BglII fragment K appear to be 0.3-kb shorter than their corresponding Se-US fragments and map to the same region, between 7 and 10 map units (m.u.), suggesting that the DNA of Se-SP2 is 0.3 kb shorter in this region than the DNA of Se-US. Se-SP2 XbaI fragment Q is about 0.1 kb longer than its equivalent Se-US XbaI fragment Q. With BamHI, the only difference observed was an additional restriction enzyme site appearing at 25 m.u. which resulted in BamHI fragments F and H in Se-SP2, which corresponds to BamHI fragment F of Se-US. Polymorphic fragments, considered suitable diagnostic markers, represent an addition or deletion of a restriction enzyme site and are shown in Fig. 1b. Viral progeny in S. exigua coinfected larvae. OBs were collected from individual cadavers after double infection of S. exigua with Se-US and Se-SP2, and the resultant viral DNA was analyzed with BglII. Virus from four of nine individual coinfected larvae showed BglII restriction enzyme patterns with several submolar bands which coincided with those that are unique for each strain (Fig. 2). Those bands that were common to both strains gave a much higher relative intensity than the polymorphic bands. These two features indicated the presence of a mixture of at least both parental DNAs in the profiles (Fig. 2, lanes 5, 6, 7, and 9). However, there was also a novel submolar band of 10.6 kb (Fig. 2, lanes 5, 6, 7, and 9) that was not present in either of the original strains (Fig. 2, lanes 1 and 11), which is suggestive of the emergence of a new, apparently recombinant, genotype. Progeny OBs recovered from one larva in which the 10.6-kb novel submolar fragment was observed were used as inocula to infect S. exigua L4 instars through several successive larval passages. The analysis of viral DNA from one larval cadaver at the third passage of virus originating from the larva in lane 9 of Fig. 2 revealed a profile in which the 10.6-kb fragment was equimolar to the other


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FIG. 4. Hybridization of Se-SUR1 BglII fragment F to BglII-digested DNA from Se-US, Se-SUR1 at passage 3, and Se-SP2. The ethidium bromide-stained lane is on the left, and the autoradiogram is on the right for each strain. Hybridization occurs with Se-SP2 BglII fragment A, Se-SUR1 BglII fragment F, and Se-SP2 BglII fragment F.

fragment F (Fig. 4). From the presence of fragments on the Se-SUR1 genome diagnostic for either parent and their relative map positions in the parent, it was possible to construct a physical map for Se-SUR1 for all four enzymes (Fig. 1c). The Se-SUR1 virus was detected early after coinfection and became dominant after passage in larvae, suggesting that it has a replicative advantage. The biological activities of Se-US, Se-SP2, and Se-SUR1 were, therefore, compared in terms of LD50 and LT50. Since no significant differences were observed between replications, the duplicate bioassays were combined in the probit analysis shown in Table 1. The LD50 values ranged from 9.2 OBs/larva for Se-SP2 to 14.8 OBs/larva for Se-US, but these were not significantly different from each other and were similar to those obtained in previous studies for the parental viruses (6, 26). The LT50 values were calculated by the equation of Biever and Hostetter (1) and differed slightly for the 27-OBs/ml dose, with Se-SUR1 showing the lowest value at 74.8 h, Se-US showing the highest value at 79.9 h, and Se-SP2 with an intermediate value at 76.0 h.

FIG. 3. BglII DNA profiles for passaged virus. Lanes: 1, Se-US; 2, Se-SUR1 after the fifth passage in larvae; 3, a mixture of Se-US, Se-SP2, and Se-SUR1 from the first passage of a mixed infection; 4, Se-SP2. An arrow indicates the novel 10.6-kb BglII F fragment in Se-SUR1. Restriction fragments of Se-US and Se-SP2 are lettered in order of size, and the sizes in kilobases of some of these fragments are also indicated.

fragments. This profile remained unchanged up to the sixth passage, indicating the stability of the new genotype (Fig. 3, lane 2). This new genotypic variant was named SeMNPVSUR1 (Se-SUR1) for Se SP2-US recombinant. In successive passages of virus from other cadavers showing evidence of the Se-SUR1 genotype as well as both parental viruses (Fig. 2, lanes 5 to 7), the Se-SUR1 genotype was retained along with the parental viruses for at least two to three passages. In those cases, the replacement of parental viruses took longer. Description of Se-SUR1. Analysis of the Se-SUR1 genotype for fragments diagnostic for either the Se-US or Se-SP2 strain indicated that parts of the Se-SUR1 genome were derived from the Se-SP2 strain and that others were derived from the Se-US strain, suggesting that recombination between these strains had occurred. The Se-SUR1 BglII C and E fragments appeared to be derived from the Se-SP2 BglII C and E fragments, respectively, while the Se-SUR1 BglII M and N fragments were derived from the Se-US parent BglII K and L fragments, respectively. The diagnostic BamHI, PstI, and XbaI fragments in Se-SUR1 all seemed to be derived from the Se-SP2 parent. The Se-SUR1 BamHI F and H fragments were equivalent to the Se-SP2 BamHI F and H fragments, respectively. The Se-SUR1 PstI H fragment was equivalent to the Se-SP2 PstI H fragment. Se-SUR1 XbaI fragments Q, O, and R were equivalent to Se-SP2 XbaI fragments Q, R, and O, respectively. In addition to fragments diagnostic for either parent, the Se-SUR1 genome has a novel 10.6-kb BglII fragment absent from both parents. This DNA fragment hybridized strongly to parental Se-US BglII fragment A, Se-SP2 BglII fragment F, Se-SP2 BglII fragment P, and, as expected, to Se-SUR1 BglII

DISCUSSION The possibility that newly introduced baculoviruses might recombine with indigenous strains and produce recombinants with novel characteristics is an important consideration for the introduction of not only genetically engineered baculoviruses but also novel, perhaps geographically distinct strains as well. The results of the present study show that Se-US and Se-SP2 can recombine when they coinfect the same insect host. The recombinant Se-SUR1 which resulted from such a recombination event showed up independently in several larvae as early as within one round of coinfection. Moreover, it replaced the parental viruses after three successive passages in S. exigua larvae and appeared to be stable and dominant at least up to the sixth passage. Our observations are similar to those of others describing a high rate of recombination after the first passage in vitro (10, 21, 23, 28) as well as in vivo (8, 9, 11). In contrast to our approach using per os infection with OBs for coinfection and serial passage in the larval host, others estab-

TABLE 1. Dosage mortality and relative potency of Se-SUR1 and its parental strains Se-US and Se-SP2 for second-instar S. exigua larvae SeMNPV strain

Se-US Se-SP2 Se-SUR1

Regression line

y 5 1.69x 1 3.03 y 5 1.69x 1 3.38 y 5 1.69x 1 3.31

LD50 fiducial limits (95%)

LD50 (OBs/larva)

14.8 9.2 10.1

Relative potency

Upper

Lower

11.6 7.3 8.3

19.3 11.7 12.3

1.00 1.62 1.47

Potency fiducial limits (95%) Upper

Lower

1.15 1.08

2.28 2.02

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lished coinfections in larvae by injecting DNA (9) or extracellular viruses (8, 11) into the hemocoel. Infection of larvae through contaminated diet more closely mimics what might happen under natural conditions. Per os infections would impose a strong host selection pressure for the viruses and their progeny, and therefore only the most highly competitive recombinants would survive upon serial passage under similar experimental or field conditions. In our study, other recombinants may have arisen but, unlike Se-SUR1, may not have been sufficiently competitive and therefore did not survive selection. A similar suggestion was raised by Croizier et al. (9) in their analysis of viral progeny from double infections in Galleria mellonella larvae. The identity and relative location of diagnostic markers from Se-US and Se-SP2 that were found in Se-SUR1 suggest that Se-SUR1 was generated as a result of recombination involving at least four crossovers. These putative crossover sites would map between 29 and 38, 41 and 53, 60 and 88, and 91 and 4 m.u. as shown in Fig. 1d. The crossovers could not be mapped more precisely because of the sizes of common fragments and lack of marker fragments in some regions of the genome. The first crossover at 29.5 to 38.2 m.u., where Se-US BglII fragment A and Se-SP2 BglII fragment F are involved, would explain the appearance of the unique Se-SUR1 BglII F fragment. This would contain the right end sequence derived from the Se-US BglII A fragment and the left end sequence of the Se-SP2 BglII F fragment. This is also supported by the absence in Se-SUR1 of a 0.6-kb Se-SUR1 BglII P fragment. The evidence for the second, third, and fourth crossovers is indicated by the presence on the Se-SUR1 genome of the Se-US BglII L marker (Se-SUR1 BglII N) between 38.2 and 41.0 m.u., the Se-SP2 PstI H (Se-SUR1 PstI H) and Se-SP2 XbaI O and R (Se-SUR1 XbaI O and R, respectively) markers between 53.7 and 59.6 m.u., and the Se-US BglII K marker (Se-SUR1 BglII M) between 88.0 and 91.2 m.u., respectively. Further characterization of the recombinant genome including the sequencing of the 10.6-kb BglII fragment could provide an additional explanation for the observed replacement. The biological activity of Se-SUR1 in terms of either LD50 or LT50 are not significantly different from that of the parental viruses. The differences between the LT50 values for the parental strains and those of Se-SUR1 are too small to explain the replacement of the parental viruses by the recombinant one upon successive passage in vivo, although it cannot be ruled out as an explanation. Although our results strongly suggest that the Se-SUR1 that survived our selection procedure resulted from a recombination between the parental viruses in the natural hosts, it is also possible that it may already have been present as a minor variant in one of the preparations. The parental viruses used for the coinfections were not cloned; each of them was a field isolate showing heterogeneity in the DNA profiles. However, it is unlikely that Se-SUR1 was present as an occult genotype within one of the parental strains, since a polymorphic marker fragment such as the 10.6-kb BglII fragment was not observed, even by hybridization. The fact that Se-SUR1 is readily detectable after the initial coinfection in four of nine larvae, along with the distribution of DNA markers from two geographical distinct parents, and also that Se-SUR1 was not among the different Se-SP2 genotypes obtained in vivo cloning (24a) argue in favor of recombination. In control experiments with larvae infected by either Se-US or Se-SP2, viral DNA patterns showed no change with passage and no pattern representative of Se-SUR1 was observed. Viral mortality was also not detected in mock-infected larvae, indicating that Se-SUR1 could not have been a latent contaminant in the larval population.


Finally, despite routine culture of SeMNPV by repeated passage in S. exigua in our laboratory, we have never observed any changes in the REN profiles of the virus. Nevertheless, PCR analysis using oligonucleotide primers designed to amplify a Se-SUR1-specific region and used with template DNA from Se-US and Se-SP2 preparations could be used to rule out the possibility of the presence of such a rare genotype. Due to the recombinant nature of baculoviruses, Kondo and Maeda (21) also suggested recombination as one of the mechanisms involved in host range expansion of these viruses. Recombination may play an important role in the generation of variability between baculovirus strains too, as demonstrated in the case of AgMNPV (10). For SeMNPV, several strains have been isolated and characterized in different geographical regions of the world, namely, three in Spain (two of these have been described [6], and the third one is unpublished), five in Japan (15, 22), one in Thailand (6), and at least two in the United States (14). However, the contribution of recombination events to SeMNPV diversity still needs to be evaluated. The results reported here add further evidence to the high frequency of recombination among baculoviruses. Our demonstration here that this can also occur in the natural host indicates that (i) introduction of foreign strains of SeMNPV in regions where natural strains already exist may result in the appearance of new genotypes from recombination between the parental viruses, (ii) the recombinants may replace the parental strains, and (iii) the recombinants may have an altered biological activity. For all these reasons, epidemiological and ecological studies are strongly recommended before the release of new baculovirus insecticides with active components different from the natural population strains. It is also recommended that extensive field sampling be carried out prior to releasing new strains to preserve the natural diversity of baculovirus strains. These considerations should also be taken into account, especially before genetically engineered recombinants are released. Those carrying a deletion might be preferable to those expressing a heterologous gene since homologous recombination might transfer the nonbaculovirus gene to the genome of indigenous field strains which may outcompete other baculoviruses in the field. ACKNOWLEDGMENTS We thank Peter C. G. Krell for critically reading the manuscript. Delia Mun õz is the recipient of a grant from the regional government of Navarra, Spain. REFERENCES 1. Biever, K. D., and D. L. Hostetter. 1971. Activity of the nuclear polyhedrosis virus of the cabbage looper evaluated at programmed temperature regimens. J. Invertebr. Pathol. 18:81–84. 2. Brewer, M. J., and J. T. Trumble. 1989. Field monitoring for insecticide resistance in beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 82:1520–1526. 3. Brooks, W. M. 1993. Host-parasitoid-pathogen interactions, p. 231–272. In N. E. Beckage, S. N. Thompson, and B. A. Federici (ed.), Parasites and pathogens of insects, vol. 2. Academic Press, Inc., San Diego, Calif. 4. Brown, E. S., and C. F. Delhurst. 1975. The genus Spodoptera (Lepidoptera: Noctuidae) in Africa and the Near East. Bull. Entomol. Res. 65:221–262. 5. Caballero, P., H. K. Aldebis, E. Vargas-Osuna, and C. Santiago-Alvarez. 1992. Epizootics caused by a nuclear polyhedrosis virus in populations of Spodoptera exigua in southern Spain. Biocontrol Sci. Technol. 2:35–38. 6. Caballero, P., D. Zuidema, C. Santiago-Alvarez, and J. M. Vlak. 1992. Biochemical and biological characterization of four isolates of Spodoptera exigua nuclear polyhedrosis virus. Biocontrol Sci. Technol. 2:145–157. 7. Chaufaux, J., and P. Ferron. 1986. Sensibilite´ diffe´rente de deux populations de Spodoptera exigua Hub (Lep., Noctuidae) aux baculovirus et aux piretroides de synthe`se. Agronomie 6:99. 8. Croizier, G., D. Godse, and J. M. Vlak. 1980. Se´lection des types viraux dans les infections doubles à Baculovirus chez les larves de Le´pidopte`re. C. R. Acad. Sci. Paris, t290:23–25.

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9. Croizier, G., L. Croizier, J. M. Quiot, and D. Lereclus. 1988. Recombination of Autographa californica and Rachiplusia ou nuclear polyhedrosis viruses in Galleria mellonella L. J. Gen. Virol. 69:177–185. 10. Croizier, G., and H. C. P. Riveiro. 1992. Recombination as a possible major cause of genetic heterogeneity in Anticarsia gemmatalis nuclear polyhedrosis virus populations. Virus Res. 26:183–196. 11. Croizier, G., and J. M. Quiot. 1981. Obtention and analysis of two genetic recombinants of baculoviruses of lepidoptera, Autographa californica (Speyer) and Galleria mellonella L. Ann. Virol. 132E:1–18. 12. Finney, L. 1971. Probit analysis. Cambridge University Press, London, United Kingdom. 13. Gelernter, W. D., N. C. Toscano, K. Kido, and B. A. Federici. 1986. Comparison of a nuclear polyhedrosis virus and chemical insecticides for control of the beet armyworm (Lepidoptera: Noctuidae) on head lettuce. J. Econ. Entomol. 79:714–717. 14. Gelernter, W. D., and B. A. Federici. 1986. Isolation, identification and determination of virulence of a nuclear polyhedrosis virus from the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). Environ. Entomol. 15:240–245. 15. Hara, K., M. Funakoshi, and T. Kawarabata. 1995. In vivo and in vitro characterization of several isolates of Spodoptera exigua nuclear polyhedrosis virus. Acta Virol. 39:215–222. 16. Hartig, P. C., M. C. Cardon, and C. Y. Kawanishi. 1991. Insect viruses: assays for viral replication and persistence in mammalian cells. J. Virol. Methods 31:335–344. 17. Heldens, J. G. M., E. A. van Strien, A. M. Feldmann, P. Kulcsar, D. Mun ˜ oz, D. J. Leisy, D. Zuidema, R. W. Goldbach, and J. M. Vlak. 1996. Spodoptera exigua multiple Nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo. J. Gen. Virol. 77:3127–3134. 18. Huber, J. 1986. Use of baculoviruses in pest management programs, p. 181–203. In R. R. Granados and B. A. Federici (ed.), The biology of baculoviruses, vol. 2. CRC Press, Inc., Boca Raton, Fla. 19. Hughes, P. R., and H. A. Wood. 1981. A synchronous peroral technique for the bioassay of insect viruses. J. Invertebr. Pathol. 37:154–159. 20. Kolodny-Hirsch, D. M., D. L. Warkentin, B. Alvarado-Rodrı´guez, and R. Kirkland. 1993. Spodoptera exigua nuclear polyhedrosis virus as a candidate viral insecticide for the beet armyworm (Lepidoptera: Noctuidae).

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J. Econ. Entomol. 86:314–321. 21. Kondo, A., and S. Maeda. 1991. Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus. J. Virol. 65:3625–3632. 22. Kondo, A., M. Yamamoto, S. Takashi, and S. Maeda. 1994. Isolation and characterization of nuclear polyhedrosis viruses from the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) found in Shiga, Japan. Appl. Entomol. Zool. 29:105–111. 23. Lee, H. H., and L. K. Miller. 1979. Isolation, complementation and variants of Autographa californica nuclear polyhedrosis virus. J. Virol. 27:754–767. 24. Martignoni, M. E., and P. J. Iwai. 1981. A catalog of viral diseases of insects, mites and ticks. USDA Forest Service report GTR PNWW-195. U.S. Department of Agriculture, Washington, D.C. 24a.Mun ˜ oz, D., et al. Unpublished data. 25. Payne, C. C. 1988. Pathogens for the control of insects: where next? Phil. Trans. R. Soc. Lond. B 318:225–248. 26. Smits, P. H., and J. M. Vlak. 1988. Biological activity of Spodoptera exigua nuclear polyhedrosis virus against S. exigua larvae. J. Invertebr. Pathol. 51: 107–114. 27. Smits, P. H., M. van de Vrie, and J. M. Vlak. 1987. Nuclear polyhedrosis virus for control of Spodoptera exigua larvae in glasshouse crops. Entomol. Exp. Appl. 43:73–80. 28. Summers, M. D., G. E. Smith, J. D. Knell, and J. P. Burand. 1980. Physical maps of Autographa californica and Rachiplusia ou nuclear polyhedrosis virus recombinants. J. Virol. 34:693–703. 29. Tanada, Y., and J. R. Fuxa. 1987. The pathogen population, p. 113–159. In J. R. Fuxa and Y. Tanada (ed.), Epizootiology of insect diseases. John Wiley & Sons, Inc., New York, N.Y. 30. van Laecke, K., and D. Degheele. 1991. Detoxification of diflubenzuron in the larvae of the beet armyworm (Spodoptera exigua) (Lepidoptera: Noctuidae). Pestic. Biochem. Physiol. 40:181–190. 31. van Strien, E. A., D. Zuidema, R. W. Goldbach, and J. M. Vlak. 1992. Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus. J. Gen. Virol. 73:2813–2821. 32. Vlak, J. M., K. van Frankenhuyzen, and D. Peters. 1981. Identification of a new nuclear polyhedrosis virus from Spodoptera exigua. J. Invertebr. Pathol. 38:297–298.