A gypsy moth ( Lymantria dispar . , Lepidoptera : Lymantriidae ) multinucleocapsid nuclear polyhedrosis virus from France : comparison with a North American and a Korean strain

As part of a search for natural enemies of the gypsy moth (Lymantria dispar), virus-infected samples were collected near Toulouse, France. Light and electron microscope studies confirmed that the French strain is a multinucleocapsid nuclear polyhe­ drosis virus (MNPV). In vivo bioassays using the New Jersey strain of L. dispar, and comparing L. dispar MNPV (LdMNPV) strains from France, North America and Korea, showed that the French strain was the least active, whereas the North American strain had the highest activity. The viral efficacy of all strains was enhanced 200 to 1300-fold in the presence of 1% fluorescent brightener. The enhancement was highest in the American strain and lowest in the French strain. French LdMNPV (LdMNPVF) DNA cut with four restriction enzymes (BamHI, EcoRI, HindIII, and NotI) revealed minor fragment size differences, but many similarities when com­ pared to the North American and the Korean strain. PCR amplification of a LdMNPV early gene (G22) was detected in the North American and the Korean strain, but not in the French strain.

In June of 1998 virus-infected samples of L. dispar larvae were found in an oak forest around Pujaudran, 20 km west of Toulouse, France, in a population that was increasing 5 years after a previous outbreak.In this area, several portions of the Foret de Bouconne had suffered gypsy moth attacks of various intensities between 1990 and 1993 (Hett, 1995).Our study was designed to com pare the virulence of the French virus to other L. dispar strains from North America and Asia (Korea), as well as to partially characterize the French virus.These studies will increase knowledge concerning geographical varia tion in LdMNPV and may eventually help to select the most virulent strain for the biological control of gypsy moth.

Specimen collection
Gypsy moth larvae were collected in an oak forest [Quercus robur L. and Quercus petraea (Mattuschka) Lieblein] near Pujaudran (Gers), 20 km west of Toulouse (Haute-Garonne), France in June of 1998.Specimens were collected on tree trunks and on foliage at chest height and placed individually in 116 ml plastic cups on simplified high wheat germ diet (Bell et al., 1981).Insects were maintained in the laboratory at 24 ± 1°C, 70 ± 10% RH, and a photoperiod of 18L : 6D until death, or the * Address correspondence to: Dr. Fernando E. Vega, USDA, ARS, Insect Biocontrol Laboratory, Bldg.011A, Room 214, BARC-West, Beltsville, Maryland 20705, USA; e-mail: vegaf@ba.ars.usda.govemergence of a parasitoid or a moth.The number of larvae col lected were 1,101 on June 14 and 1,383 on June 21, 1998.

Virus extraction
The presence of occlusion bodies (OBs) in dead larvae sus pected to be virus-infected was confirmed by light microscopy.For comparative purposes, a North American strain, i.e. the Abington strain of LdMNPV (LdMNPV-Ab-a624; henceforth referred to as a624; Lynn et al., 1993) and a Korean LdMNPV strain (LdMNPVK; provided by M. Shapiro, USDA) were used.Tompkins (1991) method with minor modifications was used for viral purification.In summary, the virus-infected L. dispar cadavers were blended in a homogenizer (Virtis Research Equipment, Gardiner, New York, USA) with distilled water and strained through four layers of cheese cloth.All of the centrifu gations were performed in a refrigerated Sorvall-RC-5B centri fuge (Sorvall Products, L.P., Newtown, Connecticut, USA) using a SS-34 rotor (DuPont Instruments, Wilmington, Dela ware, USA).The mixture was centrifuged at 3000 rpm for 15 minutes.The pellet was re-suspended in distilled water and cen trifuged again at 3000 rpm for 15 minutes followed by re suspension in 10 ml of distilled water and sonicated (Branson Sonifier, VWR Scientific, Westchester, Pennsylvania, USA) at the medium setting.The suspension was then layered on a bed of 40% (w/w) sucrose solution and centrifuged at 8500 rpm for one hour.The pellet was suspended and sonicated again and layered on a 40-65% sucrose gradient and centrifuged at 20,000 rpm for 60 minutes.OBs were collected and washed in 1% SDS and 0.5 mg/ml proteinase K for 2-3 hours at room temperature.The pellet containing OBs was washed with distilled water three times and suspended in a small volume of distilled water; OBs were visually counted using a Neubauer hemacytometer.
Electron microscopy Adams et al. (1977) methodology for OB's preparation and examination was followed.In summary, the OBs were fixed in sodium cacodylate buffer, pH 7.2 (0.05 M sodium cacodylate, 0.5 mM HCl) with 0.17 M sucrose and 2.5% glutaraldehyde at 4°C overnight.The samples were washed three times in 0.05 M sodium cacodylate buffer with 0.34 M sucrose at 4°C followed by fixation in 1% osmium tetroxide at 4°C for two hours and three washes with sodium cacodylate buffer without sucrose.Samples were stained with 2% uranyl acetate (aqueous) at 4°C for 45 minutes and rinsed in sodium cacodylate buffer followed by centrifugation at 3,000g for 5 minutes.The pellet was then dehydrated through a series of alcohol concentrations (30%, 50%, 70%, 95% and 100%) for 15 minutes in each and then cut into small pieces and infiltrated in a propylene oxide:epon 812 mixture (1:1) for one hour followed by infiltration with pro pylene oxide and epon 812 (1 : 3) mixture.The samples were finally infiltered in epon 812 for 8 hours and then incubated at 60°C overnight.Thin sections were cut using a diamond knife and an LKB ultra microtome (LKB Produkter AB, Bromma, Sweden).The grids were stained with 2% uranyl acetate and lead citrate, examined using a Philips 400 T electron microscope (FEI Co., Hillsboro, Oregon, USA) and photographed.A sample of 200 cross sections of OBs from each of the three strains were photographed.The diameter of each OB cross section was measured and the number of virions in each cross section was counted.

In vivo bioassays
The three virus strains were fed to the New Jersey strain of L. dispar (USDA-APHIS, Otis Air National Guard Base, Massa chusetts, USA) and the virus extracted (using Tompkins method (1991) as previously described) for use in bioassays.The viru lence (measured as the virus amount required for 50% effective lethal concentration, or LC50) of the three LdMNPV strains was determined using the techniques of Shapiro & Argauer (1995).The New Jersey strain of L. dispar was used for bioassays.
To test whether fluorescent brighteners would enhance viral activity, as has been previously reported (Shapiro & Robertson, 1992;Dougherty et al. 1995), serial dilutions of eight concentra tions of the stock OBs suspensions were prepared either in dis tilled water or in 1% (wt/wt) fluorescent brightener 28 (Calcofluor white M2R, Sigma Chemical Co., St. Louis, Mis souri, USA).One milliliter of the viral suspensions was applied to the diet surfaces (90 ml of diet in a 180 ml cup) at concentra tions ranging from 101 to 108 OBs/ml per cup.Each diet cup contained ten 2nd instar larvae.Two cups of controls were used in each set, one containing water and the other 1% (wt/wt) fluo rescent brightener 28.Tests were repeated six times for each concentration, with three replicates per treatment each time.Larvae were reared in a growth chamber at 29 ± 2°C under a 12L : 12D cycle at 50% RH and were observed from day 1 to day 14.Dead larvae were counted and dosage-mortality data was determined using Probit analysis (Russell et al., 1977).

Molecular analyses Extraction of DNA from OBs
Viral DNA was extracted using Loh's et al. (1981) procedure.The OB's were re-suspended in an extraction buffer consisting of 0.14 M NaCl, 0.03 M KCl, 0.01 M N 2HP04 , 2mM KH2PO4 (pH 7.4), 1% SDS, 0.08 M EDTA (pH 8.0) The proteins were digested by adding 500 pg/ml of proteinase K and incubated for one hour at 37°C.Na2CO3 (pH 10.6) was added to a final con centration of 0.1M, and the solution was then incubated at 37 °C for one hour.The DNA was extracted with 1 volume buffered phenol, 2 volumes of chloroform:isoamyl alcohol (24 : 1), and then precipitated with 2 volumes of cold ethanol.The precipitate was dissolved in 1 mM Tris, 0.01mM EDTA buffer (pH 7.5).

Analysis of virus DNA with restriction endonucleases
The viral DNA was digested with four restriction enzymes (BamHI, EcoRI, HindIII and Not!) using standard protocols (Miller & Dawes, 1978 ;Sambrook et al., 1989) at 37°C for two hours.The samples were then analyzed by electrophoresis in a 0.8% agarose gel and stained with 0.5 pg/ml ethidium bromide.The DNA bands were visualized using a UV transilluminator.

PCR amplification using early gene (G22) primers
The forward primer (5' CGC ACC GCG TCC ACG ATA ACG AGC GC 3') homologous to nucleotides 191-216 and reverse primer (5' ACA TGG TCG TGC CCG ACC AGT CGT AA 3') complementary to nucleotides 716-741 were designed from a published sequence of an early gene (G22) in LdMNPV (Bischoff & Slavicek, 1995).The primers were synthesized by BRL (Gibco/BRL, Gaithersburg, Maryland, USA).The reaction was carried out in a final volume of 50pl consisting of 20 mM Tris HCl, 50 mM KCl (pH 8.0), 2.5 mM MgCh, 200 pM each of the four deoxynucleotide triphosphates, 20 pmols of each primer and 2.5 units of Taq polymerase (Gibco/BRL, Gaithersburg, Maryland, USA).The mixture was subjected to 35 cycles of amplification in a OMNI-E thermocycler (Hybaid, Middlesex, UK) as follows: denaturation at 94°C for one minute, annealing at 55°C for two minutes and extension at 72°C for three minutes.The template used was from LdMNPVF, a624, LdMNPVK, and LdMNPV-Bgal (a recombinant virus con taining a B-galactosidase gene under the control of the late polyhedrin promotor; provided by J.M. Slavicek, USDA Forest Service).The amplified product (5pl) was analyzed by electro phoresis using 0.8% agarose gel and visualized using a UV tran silluminator.
There are two morphological types of baculoviruses: a single nucleocapsid virus (SNPV), where only one nucleocapsid is found in each virion, and a multiple nucleocapsid (MNPV), in which a virion may contain numerous nucleocapsids.By employing both light and electron microscopy, our study confirms that the French strain belongs to the MNPV.The LdMNPVF strain showed characteristic polyhedral, proteinaceous inclusion bodies in the infected cells, which is a unique feature of the Baculoviridae, subgroup A (Bilimoria, 1991).Elec tron micrographs of the cross section of OBs from LdMNPVF revealed bundles of virions in the nucleocap sids (Fig. 1A).The average diameter of the cross section of OBs was 2.1 ± 0.06 pm in LdMNPVF, 1.99 ± 0.04 pm in a624 and 1.5 ± 0.06 pm in LdMNPVK (Figs 1A, B, C).The average size of the cross section of OBs of LdMNPVF was significantly larger than in LdMNPVK (p < 0.001), but there was no significant difference from a624.However, the number of virions/cross section of OBs in LdMNPVF and LdMNPVK (16.25 ± 0.9 and 16.2 ± 1.39, respectively) were significantly lower (p < 0.001) when compared to a624 (20 ± 1.34).
In the present study, the LC50 of LdMNPVF was the highest (1.5x104 ± 1797 OBs/ml) in contrast to a624, which had the lowest LC50 (1.6x103 ± 190 OBs/ml).The LC50 of LdMNPVK was 3.9x103 ± 890 OBs/ml (Fig. 2); bioassays using LdMNPVK had not been previously reported.Previous studies of comparative infectivities of LdMNPV strains from North America, Europe and Asia showed that the LC50 values varied from 1.7 x103 OBs/ml to greater than 5x106 OBs/ml (Shapiro et al., 1984;Shapiro & Dougherty, 1985).Comparison of five LdMNPV strains from France, Yugoslavia, Italy, United States and Japan showed greater than 1000-fold activity differences among the various strains (Magnoler, 1970); Shapiro et al. (1984) demonstrated 2900-fold differences in LC50 among 19 isolates from Asia, Canada, Europe and the USA.Our data is in agreement with previous results (Shapiro & Dougherty, 1985) showing less activity in European and Asian strains when compared to a624, although it should be noted that results might vary from ours when other strains of gypsy moth are used.Magnoler (1970) has also reported high activity from a USA strain when compared to strains from France and Japan.
Comparisons of restriction endonuclease fragment pro files is an important tool to identify molecular differences among various viral species and strains ( Smith & Sum mers, 1978;McCarthy et al., 1979;;Maruniak et al., 1984;Cherry & Summer, 1985;Shapiro & Dougherty, 1985;Kuzio et al., 1999).The viral genome DNA of LdMNPVF, a624, and LdMNPVK digested with BamHI, EcoRI, HindlII, and Notl showed similarities (Fig. 3) among all the major group of fragments present in all three strains.There were, however, differences in some of the fragment sizes among three strains.The general pat tern profile agrees with previous studies (McCarthy et al., 1979;McClintock & Dougherty, 1988) where LdMNPV DNA was digested with BamH1, EcoR1 and Hind III.Lane 1 is from a healthy L. dispar cell line (control).The amplified product of 524 base pairs was evident in a624 (lane 2) and LdMNPV P-gal (lane 3), whereas the LdMNPVK showed a very faint product (lane 4).No amplified product could be seen with the LdMNPVF template or in the control.Lane M is the 40 Kb marker.
However, there are more minor fragments present when the French isolate was digested with BamHl.
PCR has been useful to detect various baculoviruses (Moraes & Maruniak, 1997).Using this technique, it is possible to detect the amplified gene of interest in various viral strains and thus to distinguish similarities among various populations or strains.In the present study, a 524 base pair fragment found in an early gene (G22) of LdMNPV amplified when DNA from a624, LdMNPV-ßgal, and LdMNPVK strains were used (Fig. 4).However, there was no amplification of the early gene fragment when LdMNPVF was used, perhaps due to a primer mis match in the 3' end.Future studies using hybridization probes for this gene will determine if it is present or absent in the French strain.
Our study shows that the French isolate of LdMNPV is the least virulent among the three strains, and that fluores cent brighteners enhance viral efficacy.In addition, LdMNPVF has many similarities to North American and Korean LdMNPV strains based on restriction enzyme analysis, although there was no amplification, using PCR, of an early gene (G22) in the French strain.
Fig. 1.Electron micrograph of the cross section of several purified occlusion bodies (OBs) isolated from LdMNPVF (1A).Figs.1B and 1C show a representative number of OBs isolated from LdMNPVK and a624, respectively.

Fig. 2 .
Fig. 2. Comparison of log LC50 among LdMNPV isolates from France, Korea and North America.LdMNPVF is least active when compared to North American and Korean isolates.Note the large decrease in log LC50 of the larvae fed with LdMNPV with 1% fluorescent brightener 28 in all three viruses isolates.

Fig. 4 .
Fig. 4. PCR amplification of early gene (G22) sequence from LdMNPV using DNA templates of a624 (lane 2), LdMNPV p-gal (lane 3), LdMNPVK (lane 4) and LdMNPVF (lane 5).Lane 1 is from a healthy L. dispar cell line (control).The amplified product of 524 base pairs was evident in a624 (lane 2) and LdMNPV P-gal (lane 3), whereas the LdMNPVK showed a very faint product (lane 4).No amplified product could be seen with the LdMNPVF template or in the control.Lane M is the 40 Kb marker.