A New Nucleopolyhedrovirus Strain (Ldmnpv-Like Virus) with a Defective Fp25 Gene from Lymantria Xylina (Lepidoptera: Lymantriidae) in Taiwan

Journal of Invertebrate Pathology 102 (2009) 110–119

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Journal of Invertebrate Pathology

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A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan

Yu-Shin Nai a,1, Tai-Chuan Wang a,1, Yun-Ru Chen a,1, Chu-Fang Lo b,*, Chung-Hsiung Wang a,b,* a Department of Entomology, National Taiwan University, Taipei, Taiwan, ROC b Department of Zoology, National Taiwan University, Taipei, Taiwan, ROC article info abstract

Article history: A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, Received 1 August 2008 (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y Accepted 13 July 2009 and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The Available online 17 July 2009 restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than Keywords: that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and LyxyMNPV lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed LdMNPV-like virus that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phyloge- LdMNPV Fp25k mutant netic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally RFLP named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction chemical insecticides are still used to control L. xylina caterpil- lars, but this method causes serious environmental impacts, as The casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: well as raising human health concerns. Lymantriidae), is a herbivore that feeds on casuarina (Casuarina A nucleopolyhedrosis epizootic of L. xylina larvae occurs each equisetifolia), guava (Psidium guajava L.), lingan (Euphoria longana year from spring to early summer in Taiwan and mainland China, Lam.), lichi (lychee, Litchi chinensis Sonn.), acacia (Acacia confusa) and the key pathogen was found to be L. xylina multiple nucleo- forests and more than 60 other species of host plants (Chao polyhedrovirus (LyxyMNPV) (Cheng et al., 1987; Liang et al., et al., 1996; Shen et al., 2003; Shen et al., 2006). It is native to 1986; Wu and Wang, 2005; Xiao, 1992; Yu et al., 1997). Lyx- Taiwan, Japan, India, and the eastern coast of mainland China yMNPV’s high virulence to L. xylina suggested that it would be a (Chao et al., 1996; Matsumura, 1933; Xiao, 1992), but in Taiwan, promising agent to include in integrated pest management (IPM) in the last 30 years, as forest was completed to crops, the moth programs for the biological control of L. xylina. According to Wu expanded its range into these newly established agricultural and Wang (2005), LyxyMNPV is closely related to LdMNPV and be- areas and simultaneously expanded its host plant range (Chao longs to group II NPV. et al., 1996). Since 2001, infestations of this moth have damage In the present paper, we characterize a new Lymantria-de- to the Taiwan fruit industry, and L. xylina is now considered to rived virus that was ﬁrst isolated from infected larva of L. xylina be a signiﬁcant pest that is subject to quarantine in Taiwan (Yu et al., 1997). This new strain showed a high identity to the (Chao, 2002). L. xylina is phylogenetically closely related to the polyhedrin sequence of LdMNPV, and the in vitro infectivity of gypsy moth, Lymantria dispar, a very important forest pest in this strain in LY and LD cells was similar to that of wild-type Europe and North America (Solter and Hajek, 2009). In Taiwan, (WT) LyxyMNPV (Wu and Wang, 2005, 2006). Over 800 different baculovirus isolates from a variety of invertebrates have been reported. Genotypic variants or distinct isolates have been isolated * Corresponding authors. Fax: +886 2 27364329 (C.H. Wang). E-mail address: [email protected] (C.-H. Wang). from the same host species from sympatric or allopatric areas 1 Equal contribution to this paper. (Lee and Miller, 1978; Gettig and McCarthy, 1982; Cherry and

0022-2011/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.07.004 Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 111

Summers, 1985; Lee and Lee, 1988; Maeda et al., 1990; Slavicek (CPE) of the FP variants is usually defined as less than 10 OBs et al., 1992; Weitzman et al., 1992), whereas few polyhedra (FP) in the infected cells, FP variants not only show reduced numbers (few occlusion bodies or OBs) and many polyhedra (MP) or OB of OBs in the infected cell, but also OBs with few or no virions variants of NPVs usually occur during the serial cell culture pas- occluded. In contrast, FP variants increase the yield of budded sage at high M.O.I. (multiplicity of infection) rates (Tramper and virus (BV) (Harrison and Summers, 1995; Bull et al., 2003; Wu Vlak, 1986). et al., 2005). Since Hink and Vail (1973) first observed FP mutants in the The FP CPE is mainly the result of a deficient fp25k gene in the AcMNPV-infected Trichopusia ni cells, the common properties of baculoviral genome. The fp25k mutants usually produce a great the FP CPE have been studied in several baculoviruses. In vitro number of budded virus (BV) into the cell culture medium, and serial passage of baculovirus will frequently lead to production virus progeny at 72 h post infection (pi) are still observed budding of FP variants (Fraser et al., 1983). The cyptopathoic effects at the plasma membrane, whereas in wild-type (WT) NPVs, BV pro-

Table 1 Baculoviruses Polyhedrin, LEF-8 and LEF-9 amino acid sequences used in this study.

Group Virus name Accession number References Polyhedrin LEF-8 LEF-9 Lepidopteran NPV-I AcMNPV NC_001623 NP_054079 NP_054092 Ayres et al. (1994) AgMNPV YP_803395 YP_803445 YP_803456 Oliveira et al. (2006) AnpeNPV YP_610970 YP_611067 YP_611058 Nie et al. (2007) BmNPV NP_047414 NP_047454 NP_047466 Gomi et al. (1999) CfMNPV NP_848313 NP_848359 NP_848370 de Jong et al. (2005) CfDE FNPV NP_932610 NP_932656 NP_932667 Lauzon et al. (2005) Epp oNPV NP_203170 NP_203214 NP_203225 Hyink et al. (2002) HycuNP V YP_473189 YP_473277 YP_473277 Ikeda et at. (2006) MaviNPV YP_950731 YP_950765 YP_950776 Chen et al. (2008) OpMNPV NP_046159 NP_046210 NP_046221 Ahrens et al. (1997) RoMNPV NP_702998 NP_703040 NP_703052 Harrison and Bonning (2003) Lepidopteran NPV-II AdhoNPV NP_818648 NP_818696 NP_818682 Nakai et al. (2003) AgseNPV YP_529671 YP_529791 YP_529775 Jakubowska et al. (2006) ChchNPV YP_249605 YP_249641 YP_249656 van Oers et at. (2005) C1biNPV YP_717539 YP_717570 YP_717585 Wang et al. (unpublished) EcobNPV YP_874194 YP_874225 YP_874239 Ma et al. (2006) HearNP V (G4) NP_075070 NP_075107 NP_075124 Chen et al. (2001) HearNP V (Cl) NP_203559 NP_203594 NP_203610 Zhang et al. (2005) HzSNPV NP_542624 NP_542661 NP_542679 Chen et al. (2002) LdMNPV NP_047637 NP_047687 NP_047701 Kuzio et al. (1999) LsNPV YP_758298 YP_758340 YP_758375 Xiao and Qi (2007) LyxyMNPV AAW28 857 ACL31659 ACL31660 Wu and Wang (2005) and This study LdMNPV-like virus ACL31661 ACL31658 ACL31657 This study MacoNPV (A) NP_613084 NP_613224 NP_613207 Li et al. (2002b) MacoNPV (B) NP_689176 NP_689314 NP_689297 Li et al. (2002a) SeMNPV NP_037761 NP_037872 NP_037857 IJkel et al.(1999) SfMNP V YP_001036294 YP_001036405 YP_001036389 Harrison et al. (2008) Sp1tNPV NP_258269 NP_258306 NP_258327 Pang et al. (2001) TnSNPV YP308889 YP_308923 YP_308939 Willis et al. (2005) Lepidopteran GV CpGV NP_148785 NP_148915 NP_148901 Luque et al. (2001) Hymenopteran NPV Nea bNPV YP_667849 YP_667931 YP_667887 Duffy et al. (2006) Ne1eNPV YP_025198 YP_025279 YP_025237 Lauzon et al. (2004) NeseNPV YP_025108 YP_025188 YP_025147 Garcia-Manniak et al. (2004)

Fig. 1. Cytopathic effects (CPE) of IPLB-LD-652Y cells infected with LyxyMNPV and LdMNPV-like virus at 7 days pi showing: (A) MP (multiple polyhedra) and FP (few polyhedra) found in the LyxyMNPV-infected LD cells; (B) FP only in the LdMNPV-like virus-infected LD cells; and (C) uninfected LD cells. Bar: 25 lm. 112 Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 duction decreases to barely detectable levels (Braunagel et al., Lymantria-derived virus system will provide the in vivo and in vitro 1999; Wu et al., 2005). co-infection system to evaluate the pathogenesis of simultaneous In nature, the population of a WT genotype is highly predomi- infection in host cells. nant over that of the occlusion-negative genotype and finally the population of occlusion-negative genotype will be lost (Bull et al., 2003). In laboratory, the occlusion-negative genotype can be main- 2. Materials and methods tained in the susceptible cells. Usually, FP mutant virus has been observed to persist in a stable (Bull et al., 2003). The process of 2.1. Insect cell lines and viruses such co-occlusion with the WT and the occlusion-negative genotypes had been suggested as a strategy for application of the genet- IPLB-LD-652Y cells (L. dispar cell line; Goodwin et al., 1978) ically modification baculovirus for insecticidal purposes (Wood and NTU-LY cells (L. xylina cell line, Wu and Wang, 2006) were et al., 1994). cultured in TNM-FH medium (Hink and Strauss, 1976) contain- In this study, we characterize this isolate, designated LdMNPV- ing 50 IU/ml penicillin, 50 lg/ml streptomycin, 1.25 lg/ml fungi- like virus, and show that it is a few polyhedral (FP) strain of zone, and 8% fetal bovine serum (FBS) supplement at 28 °C. WT LdMNPV. These Lymantria-derived virus from L. xylina, LyxyMNPV LyxyMNPV was originally collected from LyxyMNPV-infected L. and LdMNPV-like virus, were also compared in present paper. This xylina larvae. For virus purification, it was extracted from exper-

Fig. 2. Ultrastructures of Lymantria-derived viruses, the LyxyMNPV and LdMNPV-like virus, infected LD cells at 7 days pi showing: (A) LyxyMNPV-infected LD cells have many polyhedra in the nucleus; (B) all these polyhedra have virions occluded; In LdMNPV-like virus-infected cells; (C) there were only a few polyhedra. These polyhedra were abnormal in morphology and were surrounded by a layer of electron-moderate material, while many nucleocapsids remained in the virogenic stroma, and the OBs contained only a few virions; and (D) no enveloped virions were occluded in the polyhedra. c: calyx; n: nucleus; emm: electron-moderate materials; nc: nucleocapsids; p: polyhedron; pm: plasma membrane; vs: virogenic stroma; v: virion. Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 113 imentally infected LD cells. LdMNPV-like virus was isolated from graphs of the infected cells were taken with a JOEL JEM-1010 trans- LyxyMNPV-infected larvae by the end point limited dilution mission electron microscope at 80 kV. method (Yu et al., 1997). 2.3. Purification of viral budded virus (BV) and extraction of viral DNA 2.2. Light and transmission electron microscope observation After 14 days pi, the LdMNPV-like virus-infected LD cells were centrifuged at 3000g. for 10 min. The supernatant was layered on LD cells (3 106) were seeded in 25T flasks and absorbed with a 20% (w/v) sucrose solution and then precipitated by centrifuga- LyxyMNPV and LdMNPV-like virus (10 MOI/each) for 1 h, respec- tion at 25,000g. for 1.5 h in a P28S rotor. The pellets were resus- tively. The infected cells were observed at 7 days pi by phase-con- pended in 1TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.6), trast light microscopy (400). The preparations for ultrastructural with a final concentration of 1% (w/v) SDS and then incubated with studies of the LdMNPV-like virus-infected cells were conducted proteinase K (0.25 mg/ml) at 56 °C for 3 h. The DNA extraction using the methods of Wu and Wang (2005). Briefly, the LdMNPV- methods followed previous methods (Chou et al., 1996; Wang like virus-infected LD cells at 7 days pi were scraped from the et al., 1996). The purification of OBs and DNA extraction of Lyx- 25T flask and centrifuged at 80g (Hettich Universal 30F/RF). The yMNPV followed the methods of Wu and Wang (2005). supernatant was discarded and the pellet of infected cells was fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C for 2 h. The sample was then 2.4. Restriction endonuclease analysis and Southern blot hybridization post-fixed in 1% osmium tetroxide in the same buffer for 1 h. After washing in cold distilled water, the fixed sample was dehydrated Genomic DNA of LdMNPV-like virus was digested with BamHI, through a serial ethanol gradient, and then embedded in Epon- EcoRI, and HindIII for 16 h at 37 °C and then separated by electro- Araldite resin. A diamond knife was used to cut ultrathin sections phoresis in 0.8% agarose gels. After electrophoresis, the DNA frag- on a Reichert OMU3 Ultramicrotome. The sections were stained ments were stained with 0.3% ethidium bromide in 1TAE and with 2% aqueous uranyl acetate followed by lead citrate. Micro- photographed under UV light. LyxyMNPV and LdMNPV-like virus

Fig. 3. Restriction enzyme profiles of the LdMNPV-like virus genome. (A) Agarose gel electrophoresis of LdMNPV-like virus DNA fragments digested with BamHI, EcoRI, and HindIII (Lanes 1–3, respectively). The restriction enzyme digested DNA fragments were designated alphabetically, starting with A for the largest fragment for each enzyme digestion, and the restriction fragment sizes were estimated relative to the marker fragments of kHindIII markers. Lane M: molecular weight markers (kHindIII). (B) Estimated sizes (in kbp) of the DNA restriction fragment profiles shown in A. 114 Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 genomic DNA were also digested with BamHI, and the DNA frag- Version 3.1 (Kumar et al., 2004) applying Kimura 2-parameter ments were then transferred to Hybond-N nylon papers (Amer- model. sham Biosciences) for Southern blot hybridization. A DIG-labeled probe for polyhedrin was prepared by a PCR DIG Prob Synthesis Kit (Roche) with primer set 35/36 (Chou et al., 1996; Wang et al., 3. Results and discussion 2000) and the PCR conditions were as below: 3 min denaturation step at 94 °C, followed by 35 cycles of 94 °C for 30 s, 50 °C for 3.1. Observations and transmission electron microscopy of cytopathic 30 s, and 72 °C for 30 s, the reaction was completed with a single effects cycle of 72 °C for 5 min using a terminal cycler (AG-9600 Thermal Station, AcuGen Systems). The Southern hybridization was per- At 7 days pi, most of the LyxyMNPV-infected LD cells were formed according to the standard procedure of Sambrook et al. showing typical cytopathic effects (CPE) of MP, and only a few in- (1989) under the manufacturer’s recommended conditions fected cells that contained one to five large OBs in their nuclei (5 SSC, 1% blocking reagent, 0.1% N-laurosarcosine, and 0.02% showed FP CPE (Fig. 1A). Only FP CPE was observed in the SDS) for 16 h at 65 °C. After hybridization, the nylon membrane LdMNPV-like virus-infected cells (Fig. 1B). In ultrastructural obser- was washed twice in 2 SSC + 0.1% SDS for 10 min at room tem- vations of the WT LyxyMNPV-infected cellls, the typical MP CPE perature followed by two washes at 65 °C for 15 min each in (Fig. 2A) had OBs that contained numerous enveloped virions 0.5 SSC + 0.1% SDS. Standard chemiluminescent detection was (Fig. 2B). By contrast, OBs of the LdMNPV-like virus-infected cells performed according to the manufacturer’s instructions (Roche), showed a typical FP morphology: they had an unusually large size and the blot was exposed to X-ray film (Kodak XAR-5). and irregular shape, and contained only a few virions. Most of nucleocapsids and virions remained in the virogenic stroma that 2.5. PCR amplification, cloning and sequencing of fp25k, polyhedrin, surrounded the Obs (Fig. 2D). Consequently, most of enveloped lef-8 and lef-9 virions failed to be occluded during the crystallization of the poly-

For cloning LyxyMNPV fp25k, we used the primer set: lyx- yFP25 kEcoRI/lyxyFP25 kBamHI: 50-GGATTCCGTTAAATTATGTGA- GAATAATT-30/50-CGGGATCCCGGAATGGACACAAATTTCGATCTC-30; For LyxyNPV-2 fp25k, we used the primer set: LDFP25F/LDFP25R: 50-ATGGACATGGATTTCGATCT-30/50-TCAAATAATGTGGGAGTAAT-30. For cloning polyhedrin, the primer set, 35/36: 50-AC (CT) TA (CT) GTGTCAGACAACAAATA (CT) TACAAA30/50-GG (CT) GCGTC (GT) GG (CT) GCAAA (CT) TT (CT) TT (AT) AC (CT) TT (AG) AA-30, was used. (Chou et al., 1996; Wang et al., 2000). For cloning lef-8 and lef-9, two speciﬁc primer sets were de- signed: lef-8-f/lef-8-r: 50-ACAAGATCATCTACAAGCACG-30/50-TC AAGTGCGGAATGTAGG-30; lef-9-f, 50- CAACGAGCACACGGACA-30/ lef-9-r, 50-CCGACTTTGACCCTGTT-30. All the PCR products were cloned into T&A cloning vector (RBC Bioscience) and sequenced commercially (Mission Biotech, Taiwan) using the ABI PRISM Dye-Terminator cycle sequencing ready reaction kit and an ABI 377 automated sequencer (Perkin–Elmer).

2.6. Nucleotide sequence analysis and comparison

The nucleotide and amino acid sequences of fp25k, polyhedrin, lef-8, and lef-9 amplicons were pre-processed and assembled by the computer program Genework, Version 2.5.1. The DNA and amino acid sequences of LdMNPV-like virus were then compared with the LyxyMNPV and LdMNPV (Table 1) from the latest GenBank dat- abases (NCBI network) using BLAST (Altschul et al., 1990). Multiple sequences were aligned using CLUSTAL_X, Version 1.81 (Thompson et al., 1997). Pairwise identity and similarity were calculated by GeneDoc (Nicholas and Nicholas, 1997) with a score table of Blo- sum 35.

2.7. Phylogenetic analysis

Phylogenetic trees were inferred from a data set of combined partial amino acid sequences of the polyhedrin, lef-8, and lef-9 genes (Lange et al., 2004; Jehle et al., 2006) of 33 baculoviruses, Fig. 4. Genomic DNA of LyxyMNPV and LdMNPV-like virus (Lanes 1 and 2, see Table 1. Neighbour-joining (NJ) and maximum parsimony respectively) digested with BamHI restriction endonuclease and localization of (MP) analyses were performed using MEGA, Version 3.1 (Kumar polyhedrin by Southern blot hybridization. (A) Agarose gel electrophoresis of et al., 2004). CpGV (Luque et al., 2001) was selected as the out- LyxyMNPV and LdMNPV-like virus DNA fragments digested with BamHI. Lane M: group. Bootstrap analyses were performed to evaluate the robust- molecular weight markers (kHindIII). (B) Southern blot hybridization using a probe of a LdMNPV-like virus polyhedrin fragment amplified by DIG-PCR with a primer set ness of the phylogenies using 100 replicates for NJ and MP analy- 35/36 showing polyhedrin localized in BamHI-D (19.5 kbp) and -H (7.9 kbp) ses. Distance matrices from aligned nucleotide sequences were fragments of LyxyMNPV (Lane 1) and in BamHI-A (22.3 kbp) fragment of LdMNPV- determined by using the Pairwise Distance calculation of MEGA, like virus (Lane 2). Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 115 hedron. We also note that in the LdMNPV-like virus-infected cells, et al., 1990). FP strains are usually generated by serial passage with the polyhedra were surrounded by a thick layer of electron-moder- high MOI for several baculoviruses and the number of OBs pro- ate material (Fig. 2C). We speculate that this structure might be a duced by an FP mutant can vary according to different host cell part of a developing polyhedron or unsuccessfully crystallizing type in in vitro culture and in different tissues in infected insects polyhedron, but this will need to be further investigated in a future (Ramoska and Hink 1974; Fraser and Hink 1982; Fraser et al., study. 1983; Cusack and McCarthy, 1989; Slavicek et al., 1992; Bischoff LyxyMNPV was isolated from wild-collected NPV-infected lar- and Slavicek, 1997; Braunagel et al., 1999). We question, however, vae and LdMNPV-like virus is an FP strain also isolated from whether LdMNPV-like virus is an FP mutant from the wild-type wild-collected NPV-infected larvae. In nature, therefore, L. xylina LyxyMNPV or a distinct baculovirus coexisting in nature with Lyx- larvae consisted of at least two genetic viral variants that consisted yMNPV in the host population. of MP and FP strains. The coexistence of different genetic variants in baculovirus isolates is commonly reported, for example, geno- 3.2. Restriction endonuclease analysis and Southern blot hybridization typic variation has been reported in wild-type NPV isolates of Heliothis spp. from different geographical regions (Gettig and Restriction enzyme analysis of the LdMNPV-like virus genomic McCarthy, 1982); two distinct NPV types were isolated from Spo- DNA yielded 12 BamHI fragments; 14 EcoRI fragments; and 17 Hin- doptera littoralis (Cherry and Summers, 1985); 20 different geno- dIII fragments (Fig. 3). We compared these results to the restriction types were found in HzSNPV Klkar isolate (Corsaro and Fraser, endonuclease profiles of LyxyMNPV (Wu and Wang, 2005) and 1987); two variants are reported for PfMNPV (Panolis flammea LdMNPV (McClintock and Dougherty, 1988). A mean size of 139 MNPV) (Weitzman et al., 1992); and two LdMNPV isolates were kbp for the LdMNPV-like virus genome was estimated from the isolated from a production lot of the microbial pesticide Gypchek sum of the fragment sizes, which is far smaller than that of Lyx- (Slavicek et al., 1992). Cloned strains were examined for genetic yMNPV (154 kbp) (Wu and Wang, 2005) and LdMNPV (160 kbp; variations in WT virus stock by in vitro plaque purification (Maeda AF081810).

Table 2 Pairwise comparisons of the nucleotide and putative amino acid sequences for selected genes, fp25k, polyhedrin, lef-8 and lef-9 of three closely relative NPVsa.

Virus nameb LdMNPV-like virus LyxyMNPV LdMNPV fp25k polyhedrin lef-8 lef-9fp25k polyhedrin lef-8 1ef-9fp25k polyhedrin lef-8 lef-9 LdMNPV-like virus — — — — 79 91 90 93 93 98 99 99 LyxyMNPV 28(38) 98(99) 91(94) 95(97) — — — — 85 91 90 93 LdMNPV 29(37) 98(99) 99(99) 98(99) 84(89) 100(100) 91(95) 95(97) — — ——

a The numbers above the diagonal line deﬁned by identical sequences indicate the percent identity of the nucleotide sequences while the numbers below the diagonal line indicate the percent amino acid identity (or similarity). b The sources of the virus gene and amino acid sequences used here are given in Table 1.

Fig. 5. Comparison of fp25k DNA sequences of three closely related NPVs, LdMNPV-like virus (Gen Bank Accession No. EU934445), LyxyMNPV (Gen Bank Accession No. FJ182052) and LdMNPV. The DNA sequence alignment indicated that a region, from 141 to 186, of LdMNPV-like virus fp25k was deleted. 116 Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119

Restriction enzymes profiles have been used to identify variants in LyxyMNPV and LdMNPV-like virus genome was significantly dif- of viruses (Lee and Miller, 1978; Miller and Dawes, 1978a; Smith ferent from the LyxyMNPV and LdMNPV. LdMNPV-like virus poly- and Summers, 1978) and to distinguish between closely related hedrin gene was located in a BamHI-A fragment but LyxyMNPV viruses, such as Rachoplusia ou (RoMNPV), AcMNPV, Trichoplusia polyhedrin gene was found in two fragments, BamHI-D and H ni (TnMNPV), Galleria mellonella (GmMNPV) (Miller and Dawes, (Fig. 4B). The LdMNPV polyhedrin gene is located on the BamHI-B 1978b; Smith and Summers, 1979), and the MNPVs of Spodoptera (38.4 kbp) fragment (Smith et al., 1988). All available evidence, frugiperda (Loh et al., 1982). Even though variance was observed therefore, reveals that LdMNPV-like virus is a new viral strain with between different genotypes and phenotypes of one species of novel features than LyxyMNPV or LdMNPV. NPV, only the submolar bands showed the differences (Cory et al., 2005; Lua et al., 2002; Munˇoz et al., 1999). Moreover, Lua 3.3. Nucleotide sequence analysis and comparison et al. (2002) showed that the restriction enzyme profile of FP mutant is similar to that of MP virus. Based on the result of restriction Four genes, fp25k, polyhedrin, lef-8 and lef-9, of the two L. xylina enzyme profiles, LdMNPV-like virus appears to be a distinct bacu- baculoviruses, LyxyMNPV and LdMNPV-like virus were cloned and lovirus from LyxyMNPV (Fig. 4A). sequenced, and those of LdMNPV were obtained from GenBank. In addition to the differences discussed above, Southern blot The DNA and the putative amino acid sequences of these genes hybridization showed that the localization of the polyhedrin gene were compared (Table 2).

Fig. 6. Phylogenetic analysis of LdMNPV-like virus based on Polyhedrin, LEF-8 and -9 partial amino acid sequences by an unrooted neighbour-joining analysis, showing bootstrap values >50% for NJ and MP trees at each node (NJ/MP). Three groups, lepidopteran NPV-I, -II, and hymenopteran NPV can be divided. LdMNPV-like virus belongs to the lepidopteran NPV-II and is closely related to LdMNPV. The granulovirus genes were included as an out-group. Numbers indicate bootstrap scores converted into a percentage of the relative frequency in which bipartitions were observed. The virus strains used are listed in Table 2. Y.-S. Nai et al. / Journal of Invertebrate Pathology 102 (2009) 110–119 117

The nucleotide sequence identities of polyhedrin, lef-8, and lef-9 reported in Taiwan. In fact, the LdMNPV-like virus is a defective genes of LdMNPV-like virus were 90–93% homologous to those of type coexisting in the LyxyMNPV population in casuarinas moth LyxyMNPV, and 98–99% to those of LdMNPV. The deduced amino population. Although the origin of LdMNPV-like virus is still a acid sequences of Polyhedrin, LEF-8 and LEF-9 of LdMNPV-like question, all evidence in this study shows that it is a new LdMNPV virus were of significant similar to those of LyxyMNPV and strain in Taiwan, and it seem to be that LdMNPV and LdMNPV-like LdMNPV, with identities of 91–100% and similarities of 94–100%. virus diverged relatively recently. After alignment of fp25k gene sequences, we found 44 nucleotides deletion in the fp25k gene of LdMNPV-like virus (Fig. 5). The fp25k 4. Conclusions identity for LdMNPV-like virus with LyxyMNPV or LdMNPV fp25k was relatively lower, 79% and 93% in nucleotide sequence identity, In this present paper, we isolated an FP strain from the infected respectively, and this deletion also cause a frameshift that accounts L. xylina larvae and characterized this virus (provisionally named for the low amino acid sequence identities, 28% and 29% in amino LdMNPV-like virus). The LdMNPV-like virus-infected cells dis- acid identity, respectively (Table 2). played classic FP CPE and produced large, but few OBs (1–5 OBs/ In AcMNPV and GmMNPV, FP mutants are usually correlated cell). Ultrastructurally, most of virus particles and nucleocapsids with mutation of a 25-kDa protein for which the gene shows a surrounded the OBs and only some particles were occasionally oc- large number of insertions or deletions (0.4–2.8 kbp) within a spe- cluded. Evidence from the molecular data, as well as phylogenetic cific region between 36 and 37 map units of the viral genome (Bisc- analyses based on three gene and amino acid sequences indicated hoff and Slavicek, 1997). The fp25k of LdMNPV-like virus showed that LdMNPV-like virus is a new LdMNPV strain from Taiwan pop- 44 bps deleted from bp 141 to the downstream bp 185 (Fig. 5). This ulations of L. xylina that have a significantly smaller genomic size result suggests that fp25k gene mutant might initiate the FP phe- than LdMNPV. This is the first report of an FP mutant from the field nomenon of LdMNPV-like virus. and also LdMNPV isolate in Taiwan.

3.4. Phylogenetic analysis Acknowledgments

LdMNPV-like virus was compared to LyxyMNPV and 31 other We appreciate Dr. Leellen Solter from Illinois Asia areas Natural completely sequenced baculovirus genomes (Table 1). The phylo- History Survey for giving comments and correcting this manu- genetic trees were constructed based on three conserved baculovi- script, and the excellent technical assistance of Technology Com- ral proteins, Polyhedrin, LEF-8 and LEF-9 (Lange et al., 2004; Jehle mons, College of Life Science, NTU (Taiwan) with TEM. This work et al., 2006). The neighbor-joining (NJ) and maximum parsimony was supported by the Grant NSC-95-2313-B-002-038 from Na- (MP) trees generated similar results, the NJ tree revealed higher tional Science Council of Taiwan and the Grant 97AS-14.2.3-BQ- bootstrap values and is shown in Fig. 6. All of the lepidopteran B2 from Council of Agriculture, Executive Yuan, Republic of China. NPVs can be clearly divided into two groups based on these three genes (Groups I and II). 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