An Alphabaculovirus Isolated from Dead Lymantria Dispar Larvae

Journal of Invertebrate Pathology 139 (2016) 56–66

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Journal of Invertebrate Pathology

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An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha – An example of adaptation to a new host ⇑ Lukasz Rabalski a,1, Martyna Krejmer-Rabalska a, ,1, Iwona Skrzecz b, Bartosz Wasag c, Boguslaw Szewczyk a a Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Laboratory of Recombinant Vaccines, Abrahama Str. 58, 80-307 Gdansk, Poland b Forest Research Institute, Department of Forest Protection, Raszyn Braci Lesnej Str. 3, 05-090 Sekocin Stary, Poland c Medical University of Gdansk, Department of Biology and Genetics, Debinki Str. 1, 80-211 Gdansk, Poland article info abstract

Article history: A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was Received 2 May 2016 found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Revised 19 July 2016 Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocap- Accepted 21 July 2016 sids of LdMNPV-BNP are enveloped together in 2–26 virions embedded in occluded bodies (OBs) very Available online 22 July 2016 similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae 7 in a relatively short time (LT50 of approximately 9 days for a dose of 2 10 OBs/ml), highlighting the Keywords: possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene con- Alphabaculovirus tent (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is The gypsy moth Lymantria dispar 157,270 base pairs long and has a notably lower G + C content in comparison to other LdMNPVs Lymantria monacha (50.3% G + C content compared to an average of 57.4% among other LdMNPVs). According to our phylo- Virus adaptation genetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which Photolyase are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneﬁcial as an active component of biopesticides that can be used during forest integrated pest management. Ó 2016 Published by Elsevier Inc.

1. Introduction ilex L., Q. pubescens Willd. and elms (Ulmus spp.) (Feltwell, 1989). Again, in France in the 1990s, the gypsy moth caused signiﬁcant The gypsy moth, Lymantria dispar L., is one of the species that damage, resulting in the removal of approximately 1 million cubic causes economic damage to forests and orchards. It is a polypha- meters of oak timber (Landmann, 1999). In Serbia, the gypsy moth gous species that can feed on more than 500 species of trees, is a major pest to broadleaf forests, where outbreaks of the species shrubs and forest cover and prefers oaks (Quercus spp.), hornbeam have occurred 18 times over a 150-year period (Tabakovic-Tosic (Carpinus spp.), birch (Betula spp.), linden (Tilia spp.) and poplar et al., 2013). It has also caused heavy losses in the eastern and (Populus spp.). northern parts of Russia, where it feeds on oaks, birches and Outbreaks of gypsy moths have been observed in different parts poplars (Lipa, 1998). In Japan, L. dispar is a pest to Larix kaempferi of the world. In Europe, it causes considerable damage, mainly in (Lamb.) Carr. and Betula platyphylla Sukaczev plantations oak stands, as exempliﬁed by the massive incidence in France in (Higashiura, 1991). Mass occurrences of the gypsy moth were also the eighth decade of the XX century, where it was found on Quercus recorded in North Africa, especially in Morocco (Hamdaoui and Luciano, 1995) and Algeria (Sabrina and Luciano, 1995), where it

⇑ Corresponding author. feeds on the cork oak (Quercus suber L.) and chestnut-leaved oak E-mail addresses: [email protected] (L. Rabalski), martyna. (Quercus castaneifolia C. A. Mey). The greatest economic damage [email protected] (M. Krejmer-Rabalska), [email protected] (I. Skrzecz), caused by the gypsy moth was in the United States, where it was [email protected] (B. Wasag), [email protected] (B. Szewczyk). introduced in the late nineteenth century. In the absence of natural 1 Equal contributors. http://dx.doi.org/10.1016/j.jip.2016.07.011 0022-2011/Ó 2016 Published by Elsevier Inc. L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 57 enemies, this insect destroyed approximately 20 million hectares above, the sediment containing OBs was suspended in distilled of oak forests (McManus, 1996). water and stored at 5 °C. L. dispar multiple nucleopolyhedrovirus (LdMNPV), a baculovirus, causes epizootics in gypsy moth populations. As a consequence, 2.2. Electron microscopy – morphology this pathogen is of interest to many researchers studying the biological control of pests. Since the late 1950s, scientists The OBs suspended in distilled water were purified with a have attempted to use this virus as a biological pesticide. In sucrose density gradient (40–65% w/w) followed by centrifugation 1978, the product ‘‘Gypchek” was registered in the USA (Reardon at 100,000g for 2.5 h. Purified OBs were fixed twice, first in a 2% et al., 1996), and a similar product, ‘‘Disparvirus”, was developed mixture of glutaraldehyde and cacodylate buffer for 15 min and in Canada (Cunningham and Frankenhuyzen, 1991). then in a 2% solution of osmium tetroxide for 1.5 h. The fixed The Baculoviridae family consists of enveloped, rod-shaped OBs were dehydrated successively in ethanol, acetone, and viruses harboring covalently closed, circular, double-stranded propylene oxide and then embedded in Epon 812 (Luft, 1961). DNA. It is the largest group of viral pathogens that infects insects Epon blocks were cut on an ultramicrotome. The sections were (Blissard and Rohrmann, 1990; Lange et al., 2004). The genome given contrast with phosphotungstic acid and then viewed with sizes of its members range from 81,755 bp (as is the case with a transmission electron microscope (JEM 1200 EXII). The diameter Neodiprion lecontei nucleopolyhedrovirus (Lauzon et al., 2004)) to of 100 OBs, as well as the number of nucleocapsids, were measured 178,733 bp (as is the case with Xestia c-nigrum granulovirus from photographs (20,000 magnification) showing cross-sections (Hayakawa et al., 1999)). Family Baculoviridae is divided through OBs. into the genera Alphabaculovirus (lepidopteran-specific NPVs), Betabaculovirus (lepidopteran-specific GVs), Gammabaculovirus 2.3. Estimation of LdMNPV-BNP biological activity (hymenopteran-specific NPVs) and Deltabaculovirus (dipteran- specific baculoviruses, which has only one representative, the Culex Caterpillars were obtained from the egg masses of the L. dispar nigripalpus nucleopolyhedrovirus (CuniNPV)) (Jehle et al., 2006; New Jersey Standard Strain received from the Northeastern Center Miele et al., 2011). The alphabaculovirus genus can be further for Forest Health Research in Hamden, Connecticut, USA. At the divided into two groups, group I and group II, depending on the beginning of the experiment, the egg masses were stored in a presence or absence of BV envelope glycoprotein, gp64. Until chamber at a temperature of 5 °C and 90% RH (relative humidity) recently only the members of group I contained gp64, but lately, for 6–7 days. Then, the eggs were surface sterilized to kill potential a new betabaculovirus – DisaGV (Diatraea saccharalis gran- pathogens by submerging them in 3.8% formaldehyde for 15 min, ulovirus) was described which possesses gp64 in its genome followed by three rinses in sterile water. The eggs were put in petri (Ardisson-Araújo et al., 2016). The presence of a gene encoding dishes kept in chambers. Twenty-four hours after hatching, the the fusion protein (F) indicates affiliation with alphabaculovirus caterpillars were transferred to Petri dishes containing 5 ml of group II (Pearson et al., 2000; Wang et al., 2014). Each member medium Wheat Germ Diet (described in (Odell et al., 1985)), mod- of the Baculoviridae family encodes a set of 37 core genes that ified by our group [Table A.1 (Appendix 1)]. Incubation of sterile are thought to be present in the common ancestor genome egg masses and caterpillar rearing were carried out at a tempera- (Garavaglia et al., 2012). ture of 25 °C, 50–60% RH and a photoperiod of 16:8 (L:D) hours. In this study, we describe the genome and determine the bio- Bioassays were carried out with 2nd instar caterpillars. Five logical activity of a new isolate of baculovirus found in infected concentrations of the OBs containing 2 103,2 104,2 105, L. dispar located in the Biebrzanski National Park, Poland. We term 2 106,or2 107 in 1 ml were prepared by serial dilutions. The this new isolate of baculovirus LdMNPV-BNP. Biological assays concentrations of OBs were determined in a Thoma cell counting were conducted to assess the ability of LdMNPV-BNP to control chamber (Hunter-Fujita et al., 1998). Before infection, the caterpil- the gypsy moth. These assays included isolation of baculovirus lars were starved for 8 h and then transferred to Petri dishes con- from the tissues of gypsy moth caterpillars, morphological charac- taining 5 ml of medium, on the surface of which 50 llofOB terization, rearing of L. dispar caterpillars on a diet treated with five suspension (or 50 ll of distilled water as a negative control) was different concentrations of LdMNPV-BNP, estimation of feeding spread. After 48 h, the insects were transferred from an inoculation quantity and mortality of infected caterpillars, calculation of LC50 to a fresh diet (Magnoler, 1970). The diet was changed twice a values (virus concentration required to cause 50% mortality of week. Ten individuals were placed in each dish, and as the growth insects) and calculation of LT50 values (time required to cause of caterpillars proceeded, their number was reduced to two 50% mortality of insects). Our studies of LdMNPV-BNP also caterpillars/dish up to L4–L5 instar. included next-generation sequencing of the whole genome, data The mortality of insects was recorded daily until death or pupa- assembly, annotation and phylogenetic analysis. tion. Dead insects were inspected for the presence of OBs using a Giemsa staining technique under a phase contrast light microscope at 1000 with an oil immersion lens (Poinar and Thomas, 2012). To 2. Materials and methods distinguish the effects of virus treatment from those caused by natural factors, the mortality of infected insects was corrected 2.1. LdMNPV-BNP isolate according to Abbott’s formula, taking into account natural mortality evaluated in the control treatment (Abbott, 1987). The results

The virus was isolated from 100 dead L. dispar caterpillars col- were used to calculate LC50 and LT50 values using probit regression lected from birches (Betula spp.) and willows (Salix spp.) growing analysis performed with POLO PC software (LeOra Software, in the Biebrzanski National Park (northeast Poland, 53°35022.600N; Berkeley, California, 1987, USA). The daily quantity of insect 22°49009.800E). The caterpillars were homogenized in a small feeding was assessed based on the dry weight of feces excreted amount of distilled water for tissue maceration and to release by one living caterpillar per day (total feces from single dish were OBs. The suspension was filtered three times through a double- divided by number of caterpillars in this dish). The differences in layered muslin cloth and the filtrate was centrifuged at 1000g for the weight of feces excreted by caterpillars in all treatments were 5 min to remove tissue fragments. The supernatant was suspended evaluated by the Kruskal–Wallis test with adopted significance in distilled water and then centrifuged at 3500g for 15 min. After level of P 6 0.05, followed by post hoc test (multiple comparisons three washes with distilled water and centrifugation as described of mean ranks for all groups) to find treatment effects that 58 L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 are significantly different from each other. The Statistic v.10 For tree showing sequenced genomes of baculoviruses, only topol- (StatSoftÒ) package was used in the analysis. ogy is presented. For the tree showing phylogeny of Lymantriinae baculoviruses and photolyase relationship are drawn to scale, with 2.4. Viral DNA extraction branch lengths measured in the number of substitutions per site. Baculovirus species demarcation criteria was calculated based on Isolated polyhedra were resuspended in sterile water and fur- published guidelines using MEGA6 software (Jehle et al., 2006; ther dissolved in an alkaline solution (0.1 M Na2CO3, pH 10.0) for Tamura et al., 2013). Collinear genomic sequences from different 30 min. After visual inspection of the transparent sample, an equal baculoviruses were aligned using the Mauve algorithm imple- volume of Tris (pH 6.4) was used for neutralization. DNA extraction mented in the Geneious 9 software (Darling et al., 2004). was performed using a MagAttract HMW DNA Kit (Qiagen) according to the manufacturer’s protocol. Prior to sequencing procedure, 3. Results DNA was diluted in nuclease-free water and tested for quality by agarose electrophoresis. 3.1. Electron microscopy studies

2.5. Genome sequencing and assembly Electron microscopy (EM) analysis revealed that the shape of the OBs is typical of nucleopolyhedroviruses (NPVs). Most of the DNA sequencing was performed using a Miseq (Illumina) and OBs had irregular shapes with a cross-sectional diameter ranging v3 sequencing chemistry. For genomic library preparation, a from 1.4 to 2.7 lm(Fig. 1). The cross-sections of polyhedra showed Nextera XT DNA sample prep kit (Illumina) was used. Paired reads 2–26 embedded virions with multiple (2–6) nucleocapsids of the target size 2 300 were generated. After trimming, normal- 25–46 nm 254–310 nm in size packaged within the envelope of ization and an error correction procedure, 432,508 reads with at the virion. The EM study details are presented in Table 1. least Q30: 92.1% were used to produce single circular contig by de novo assembling using Geneious 9 (Biomatters, http://www. 3.2. Biological activity of LdMNPV-BNP geneious.com/). The mean coverage was 356.7, with a minimum of 57 and a maximum of 404. Intergenic and repeated regions with The mortality of insects on a diet treated with the highest low coverage during de novo assembling were further amplified by concentrations of LdMNPV-BNP – 2 106,2 107 OBs/ml – was PCR and confirmed by Sanger sequencing. observed to be 74% and 90% respectively after two weeks, and finally at the end of the experiment reached 97% and 100%, 2.6. Genome annotation and phylogeny respectively. Furthermore, in the groups of insects feeding on a diet treated with the lowest concentrations of pathogen The establishment of the full nucleotide sequence for LdMNPV- (2 103–2 105 OBs/ml), the mortality increased to 15–53% after BNP was followed by open reading frame (ORF) and repeated two weeks and to 42–92% after four weeks (Fig. 2). The LC value sequences prediction. Tandem Repeats Finder and PHOBOS 50 was 7 104 OBs/ml, with 95% confidence intervals ranging from software were used for finding any genomic repeated elements 2.6 104 to 1.8 105 OBs/ml and a regression coefficient (± SD) (Benson, 1999; Kraemer et al., 2009). ORFs were identified of 0.214 ± 0.435. The LT value decreased with increasing using Glimmer3 (gene model pre-computed on RefSeq LdMNPV 50 concentration of virus and ranged from over 9 days for the genome (NC_001973)) (Delcher et al., 2007), GeneMarkS (parame- highest concentration (2 107 OBs/ml) to over 16 days for the ter: intron less eukaryotic-virus) (Borodovsky and McIninch, 1993) 2 104 OBs/ml concentration (Table 2). The pathogen used in (http://linux1.softberry.com/berry.phtml?topic=virus0&group= programs&subgroup=gfindv), and tcode EMBOSS 6.5.7 (Rice et al., 2000). Putative ORFs were then annotated by searching their translated amino acid sequence in a protein database using the HMMER web server (phmmer, UniProtKB) (Finn et al., 2011). The pfam domains, signal peptides, disordered, transmembrane and coiled-coil region indications were integrated into the HMMER search, and the results were presented for all previously unknown putative ORFs (Dosztányi et al., 2005a, 2005b; Käll et al., 2004; Lupas et al., 1991). Promoter motifs were searched for in the 150 bp region upstream from first nucleotide of any predicted ORF. Early promoters were recognized when a TATA box was present, and late promoters were recognized when a DTAAG box could be found. Amino acid sequences corresponding to predicted genes were extracted and used for multiple alignment with proteins belonging to other baculoviruses. MAFFT software with Fig. 1. Electron micrographs reveals LdMNPV-BNP polyhedral occlusion bodies automatic algorithm selection was used. Files containing aligned with embedded virions containing multiple rod-shaped nucleocapsids (magnifica- sequences were exported to MEGA6 (Molecular Evolutionary tion A – 12,000; B – 20,000 (scale bars – 500 nm)). Genetics Analysis) software for phylogenetic analysis (Katoh et al., 2002; Tamura et al., 2013). The correct substitution model and its parameters for inferred evolutionary history was chosen Table 1 based on Bayesian Information Criterion and corrected Akaike Morphological properties of Lymantria dispar multiple nucleopolyhedrovirus-BNP. Information Criterion. The phylogeny was established by using Average Dimensions of Mean number ± SD the Maximum Likelihood method based on the LG + G + I + F diameter of nucleocapsid ± SD (nm) Virions on Nucleocapsids matrix model for core protein, LG + G for DNA photolyase protein OB ± SD (lm) Diameter Length cross-sections in virions and T3 + G for nucleotide trees (Le and Gascuel, 2008). The through OB percentage of 1000 bootstrap replicates in which the associated 2.02 ± 0.36 35.06 ± 5.61 288.61 ± 20.81 14.62 ± 7.29 3.96 ± 1.93 baculoviruses clustered together is shown next to the branches. L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 59

Table 3 Mean weight of feces daily excreted by Lymantria dispar infected with LdMNPV-BNP. Different letters show signiﬁcant differences (Kruskal-Wallis test, P 6 0.05).

Treatment Mean weight ± SD (mg) in following (OBs/ml) days post infection 71421 2 103 3.43 ± 0.69a 7.65 ± 0.61a 22.51 ± 1.79a 2 104 3.46 ± 0.41a 7.55 ± 0.94a 15.93 ± 1.44b 2 105 3.89 ± 0.46a 8.28 ± 0.96a 14.45 ± 1.36b 2 106 3.91 ± 0.39a 6.48 ± 0.77a 4.34 ± 0.58c 2 107 3.82 ± 0.33a 4.08 ± 0.69b 3.64 ± 0.47c Control (water) 14.19 ± 0.96b 19.28 ± 1.68c 39.48 ± 9.12d

of virus was almost 3.5-fold higher (P = 0.0002 and P = 0.0001, respectively). Among the insects treated with pathogen, the high- Fig. 2. Cumulative mortality of Lymantria dispar infected with different concentra- est quantity of feeding was observed in the group of caterpillars tions of LdMNPV-BNP. Bioassays were carried out with 2nd instar caterpillars and starved for 8 h before exposure to OBs. Fifty microliters of OB suspension containing reared on a diet with the lowest concentrations of LdMNPV-BNP 2 103,2 104,2 105,2 106,or2 107 in 1 ml (or 50 ll of distilled water as a (approximately 20 mg of feces/insect/day; P 6 0.0001). In the case control variant) were spread on 5 ml of artiﬁcial diet in Petri dish. After 48 h, of insects developing on virus free medium, the mean weight of caterpillars were placed on fresh medium, and the mortality of insects was recorded feces reached almost 40 mg/insect/day and differed from the daily until death or pupation. Dead insects were inspected for the presence of weight of feces excreted by other individuals (P 6 0.0001). OBs using a Giemsa staining technique under a phase contrast light microscope at 1000 with an oil immersion lens. 3.4. General characteristics of the LdMNPV-BNP genome

Table 2 The complete circular LdMNPV-BNP (GenBank accession num- The LT50 values for Lymantria dispar infected with different concentrations of LdMNPV-BNP. ber: KU377538) genome is 157,270 bp long (Fig. 3). It is the short- est sequence among all of the other eight genomes of baculoviruses Treatment LT50 95% confidence Regression (OBs/ml) (days) intervals (days) coefficient ± SD isolated from the gypsy moth and available in GenBank: RefSeq NC_001973 (Kuzio et al., 1999), KF695050 (Harrison et al., 2014), 2 104 16.52 16.23–16.73 5.32 ± 0.65 2 105 13.95 13.23–14.64 5.01 ± 0.52 KP027546 (Kabilov et al., 2015), KM386655 (Harrison and 2 106 12.48 12.17–12.81 5.21 ± 0.56 Rowley, 2015), KU862282 (Martemyanov et al., 2015) and 2 107 9.65 9.16–10.02 6.15 ± 0.71 KT626570, KT626571, KT626572 (Harrison et al., 2016). The G + C content in LdMNPV-BNP is equal to 50.3%, while for other LdMNPV genomes, it ranges from 57.3 to 57.5%. the lowest concentration (2 104 OBs/ml) resulted in mortality of Furthermore, the number of detected putative open reading less than 50% of insects. frames in LdMNPV-BNP (ORFs; 150 nucleotides or longer) was 154, whereas in LdMNPV (RefSeq), there are 164. According to existing conventions, the adenine in the ATG coding for methionine 3.3. The quantity of L. dispar feeding in the polyhedrin gene was assigned as the first nucleotide, and polh was used as the first ORF in the genomic sequence of LdMNPV-BNP. The daily quantity of L. dispar feeding was varied in different We have identified 80 ORFs in forward orientation and 74 ORFs in treatments of experiments (Table 3). One week after feeding on reverse. In contrast, the LdMNPV forward-to-reverse ORFs ratio is medium with virus, the mean weight of feces (easily distinguish- 84:80, while for LyxyMNPV, another alphabaculovirus isolated able from food due to rounded shape) excreted by caterpillars from the Lymantriinae family of pests, the ratio is 79:78 (Nai was similar and did not exceed 4 mg/individual/day, while the et al., 2010). mean weight of feces of insects on virus free medium was almost Among all LdMNPV-BNP ORFs found during a homologue search 3-fold higher (Kruskal-Wallis test H = 29.864, P < 0.0001). More using a hidden Markov models analysis (HMMER), three had no distinct differences in the quantity of insect feeding were recorded hits (orf7, orf72, orf154), two were found to have an e-value lower after two weeks of rearing (H = 47.807; P 6 0.0001). The lowest than 0.01 (orf53 and orf137), and 41 were assigned as genes encod- mean weight of feces (approximately 4 mg/insect/day; ing putative uncharacterized proteins. Table A.2 (Appendix 2) P = 0.0056) was noted in the case of caterpillars feeding on diet shows the features of the 154 ORFs with the full names for encoded treated with 2 107 OBs/ml. The mean weight of feces of other proteins, their domains (if found during pfam domains, signal pep- infected insects was 1.5- to 2-fold higher and was similar in all tides, transmembrane and coiled coil search), direction, nucleotide treatments (P = 0.4645). The mean weight of excrements of control position in the genome, promoter and homologues in LdMNPV, insect was the highest and differed significantly from the other LyxyMNPV and AcMNPV (Autographa californica multiple nucle- treatments (approximately 19 mg/insect/day; P = 0.0003). opolyhedrovirus, alphabaculovirus group Ia (Ayres et al., 1994)). After three weeks, the differences in feeding quantity were even All 37 core genes present in the genomes of Baculoviridae family more evident (H = 55.265; P 6 0.0001). The feeding quantity of representatives were annotated in LdMNPV-BNP (marked in blue caterpillars rearing on food treated with higher (2 106,2 107) in Fig. 3). concentrations of pathogen did not increase throughout the dura- A universal feature of many double-stranded DNA viruses that tion of the experiment. These insects consumed less diet infect eukaryotes is the fact that they have multigene families (P = 0.0006 and P = 0.0001, respectively), and the mean weight of (MGFs) containing repeated ORFs distributed within the genome. feces remained at approximately 4 mg. Whereas feeding of cater- The Baculoviridae family possesses its own group of such genes pillars on medium with lower concentrations (2 104,2 105) called baculovirus repeated open reading frame (bro). The number 60 L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66

Fig. 3. Linear map of the LdMNPV-BNP genome. Arrows represent orientation of predicted ORFs. Core genes are marked in blue, hrs – light blue, DNA photolyase – green. The numbers indicate position in the genome nucleotide sequence. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 4. Nucleotide alignment of six imperfect palindromes in homologous repeat regions (hrs). Each sequence (hr1 to hr6) represents a consensus of the sequences of all repeated imperfect palindrome found in hrs. Respectively it was 6, 4, 5, 3, 2, 5.

of bro genes differs among all of the baculoviruses, except among with the addition of the one presented in this paper), LdMNPV- representatives of the Gammbaculovirus genus, which seems to BNP can be classified as a group II alphabaculovirus due to the lack them (Bideshi et al., 2003). LdMNPV was thought to have presence of protein F. LdMNPV-BNP has a common ancestor with the highest number of bro genes (Harrison et al., 2014), as detailed LdMNPV and LyxyMNPV, but surprisingly, LdMNPV and LyxyMNPV among the baculovirus reference sequences found in the NCBI are more closely related to each other than LdMNPV is to database. In the LdMNPV-BNP genome, also high number (18) of LdMNPV-BNP. Gene-parity plot analysis against representatives these genes were found, named bro a through bro r. from the alphabaculovirus group I (AcMNPV) and group II Baculovirus genomes often contain repeats of sequences, (LdMNPV and LyxyMNPV) is shown in Fig. 6. It is evident that referred to as homologous repeat (hr) regions, that play a role as the ORFs in LdMNPV-BNP are the most similar to those in LdMNPV, origins of DNA replication and transcriptional enhancers (Lu but many differences are still observed. The whole genome align- et al., 1997). The unit repeats in the hrs consist of imperfect ment (MAUVE) (Fig. 7) of LdMNPV, LyxyMNPV and LdMNPV-BNP palindrome core sequences flanked by conserved inverted repeats show similar blocks of gene organization. In general, it can be (Nai et al., 2010). All baculoviruses that infect moths of subfamily concluded that the LdMNPV-BNP nucleotide genome structure Lymantriinae are rich in hrs (Harrison et al., 2014; Krejmer et al., differs from the other currently known baculovirus isolates found 2015). In LdMNPV-BNP six of hrs were found and compared to each in Lymantrinae. other in Fig. 4. Previous extensive phylogenetic analysis conducted on the partial lef8 gene found in LdMNPV, LyxyMNPV, Lymantria monacha 3.5. Relationship to other baculoviruses nucleopolyhedrovirus (LymoNPV) and Lymantria fumida nucleopolyhedrovirus (LyfuNPV) isolates confirmed that each group of According to the phylogenetic tree (Fig. 5), which is based on viruses isolated from one host species cluster together (Harrison amino acid sequences of proteins encoded by all core genes from et al., 2014). We broadened the analysis by using concatenated species of the Baculoviridae family for which whole genome fragments of polh, lef8 and lef9 from 7 Lymantria spp. virus sequences are available in the GenBank database (78 genomes sequences available in GenBank and LdMNPV-BNP (only those L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 61

100 AcMNPV 100 PlxyMNPV 100 RoMNPV 100 BmNPV Clade A 100 100 BomaNPV MaviMNPV ThorNPV-p2 100 100 AgNPV 100 CfDEFMNPV 100 CoveMNPV Group I EppoNPV 100 AnpeNPV 100 PhcyNPV HycuNPV Clade B 69 100 DapuNPV 100 OpMNPV 100 ChroNPV ChmuNPV 100 CfMNPV 100 100 ChocNPV 61 EupsNPV OrleNPV 100 62 EcobNPV 32 HespNPV 100 BusuNPV SujuNPV 37 100 100 68 ApciNPV LafiNPV 100 PeluSNPV Alphabaculovirus 100 ClbiNPV LdMNPV BNP LyxyMNPV 100 50 100 LdMNPV 36 ChchNPV 100 PsinSNPV TnSNPV 52 MacoNPV-B 100 HearMNPV 100 100 MabrNPV-K1 Group II MacoNPV-A 100 PealNPV 100 100 SpliNPV-II 100 SeMNPV 91 SfMNPV 100 100 AgseNPV AgipMNPV 100 100 AgseNPV-B 60 AdhoNPV 100 AdorNPV 100 SpltNPV 100 SpliNPV LeseNPV 99 HearNPV 51 HearNPV-G4 100 HearNPV-NNg1 HearSNPV-AC53 83 100 HzSNPV 100 HearGV 100 XcGV 100 PsunGV 100 SfGV SpliGV 100 PlxyGV AgseGV 82 EpapGV 75 PhopGV Betabaculovirus 96 AdorGV 100 100 CaLGV-Henan 88 ClanGV 100 CpGV 100 CrleGV 100 PrGV ChocGV 100 ClasGV 80 100 ErelGV NeseNPV NeabNPV Gammabaculovirus 100 100 NeleNPV CuniNPV Deltabaculovirus

Fig. 5. Maximum likelihood molecular phylogenetic analysis of 79 baculoviruses. Amino acid sequences of 37 core genes were analyzed. LdMNPV-BNP (marked in red) clusters with LdMNPV and LyxyMNPV in the alphabaculovirus group II. The percentage of 1000 bootstrap replicates in which the associated baculoviruses clustered together is shown next to the branches. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 62 L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66

3.6. Comparison between the genomes of LdMNPV-BNP and other LdMNPV isolates

The LdMNPV-BNP genome is 3776 base pairs shorter than that of LdMNPV. The comparison of homologous genes annotated in LdMNPV-BNP, LdMNPV, LyxyMNPV and AcMNPV is shown in Table A.2 (Appendix 2). Focusing on the differences between baculoviruses isolated from the gypsy moth, i.e., LdMNPV-BNP and LdMNPV, one can observe that the former possesses three additional genes – cg30, gp16 and bjdp (baculovirus J domain protein). Either cg30 (orf89)orgp16 (orf129) can be found in over 40 species, while bjdp (orf44, ac51 homologue) can be found in almost 20 baculovirus genomes stored in the NCBI database. The cg30 gene is known to function as a putative transcriptional regulator, but knock-out AcMNPV and BmNPV (Bombyx mori nucleopolyhedrovirus) mutants do not affect BV or ODV production, oral infectivity or survival rate of larvae in comparison to wild type baculovirus. It has been reported that cg30 possesses two functional domains, a leucine zipper and a RING finger (Ishihara et al., 2013). A pfam domain search showed that the LdMNPV-BNP orf89 (which encodes cg30) has only a RING finger domain. Glycoprotein 16 (gp16), found frequently in nucleopolyhedroviruses, is absent in granuloviruses and is considered to be nonessential. Although gp16 seems to be associated with the nucleocapsid envelope in the cytoplasm, it was not connected to either ODV or BV. The LdMNPV-BNP gp16 is 97 amino acids long. Although, the baculovirus J domain protein was not found in the first LdMNPV nucleotide sequence published (NC_001973), it can be found in other LdMNPV genomic sequences. This fact led us to recheck the finding; we have found that bjdp is also present in the reference LdMNPV sequence between Ld-orf51 and Ld-orf53 in the forward orientation. The nucleotide sequence of bjdp is almost identical among LdMNPVs (over 95% similarity), whereas bdjp in LdMNPV-BNP is only 55.3–56.4% similar to them. The BJDP has a RNA recognition motif. Due to the similarity to bacterial DnaJ/Hsp40 (heat shock protein 40), we hypothesize that it may function as a chaperone protein. Only one copy of helicase was found in the LdMNPV-BNP genome, contrary to LdMNPV, where two copies of helicase are described (Harrison and Rowley, 2015). The LdMNPV-BNP also possesses two truncated overlapping variants of ubiquitin genes: v-ubi-A (orf32, which contains 702 nucleotides) and v-ubi-B Fig. 6. Gene-parity plot analysis. Gene parity plots of LdMNPV-BNP with LdMNPV, (orf33, which contains 684 nucleotides) confirmed by Sanger LyxyMNPV and AcMNPV based on ORF order (indicated by numbers) where the polh sequencing. Both proteins have start and stop codons. Fig. 9 repre- gene is considered as the first gene. Dots indicate homologues genes in compared genomes. sents nucleotide alignment of both genes with v-ubi from LdMNPV. In addition, another interesting trait revealed in the LdMNPV- BNP genome is the orf61 that encodes a host range factor-1. Hrf-1 three genes sequences from LymoNPV are available in GenBank; is involved in regulating the baculoviral host range and infectivity. whole genome is unknown). Surprisingly, the resulting tree It was suggested that it can promote infectivity of SeMNPV (Fig. 8) shows that the LdMNPV-BNP groups not with other isolates (Spodoptera exigua multiple nucleopolyhedrovirus), HycuNPV of LdMNPV, but instead with LymoNPV. This finding is fully (Hyphantria cunea nucleopolyhedrovirus), BmNPV and AcMNPV in supported by 100% bootstrap replicates. Close genetic linkage of non-permissive L. dispar cell lines. Recombinant AcMNPV bearing single unknown LymoNPV, LyxyMNPV and LyfuNPV isolates with hrf-1 from LdMNPV exhibited increased infectivity towards all known LdMNPV was confirmed (Harrison et al., 2014). Our Helicoverpa zea and L. dispar. This gene is thought to release the results demonstrate for the first time that baculovirus isolated block of protein synthesis late in infection (Chen et al., 1998; from L. dispar caterpillars are not only very similar genetically to Ikeda et al., 2005; Ishikawa et al., 2004). Hrf-1 was initially found viruses previously found only in Lymantria monacha but probably in LdMNPV, OpMNPV (Orgyia pseudotsugata multiple nucleopolyhe- shares a common ancestor with LymoNPV. The estimated drovirus (Ahrens et al., 1997)) and DapuNPV (Dasychira pudibunda Kimura-2-parameter distance for concatenated gene sequences of NPV (Krejmer et al., 2015)). Hrf-1 is 222 amino acids long, polh, lef8 and lef9 were determined to be 0.001 substitutions/site which makes it longer than those found in the other isolates of between LdMNPV-BNP and LymoNPV, suggesting that these LdMNPV, 113aa longer than in DapuNPV and 144aa longer than viruses are isolates of the same species (Jehle et al., 2006). In con- in OpMNPV. The similarity of amino acid sequences for the hrf-1 trast, pairwise distance between LdMNPV-BNP and the LdMNPV protein among nucleopolyhedroviruses isolated from L. dispar is reference isolate was 0.168. approximately 81–83.5%. L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 63

Fig. 7. Mauve (multiple alignment of conserved genomic sequence with rearrangements) representation of LdMNPV-BNP, LdMNPV from the NCBI database and LyxyMNPV. Alignment was performed on collinear sequences where LdMNPV-BNP was a reference sequence and the polh gene was considered as the ﬁrst ORF. The colored sections (bordered with a curve that indicates the level of nucleotide similarity) represent homologous fragments of compared genomes. The section that is located beneath the X-axis shows inversion of this genome fragment in comparison to the reference. The genomes presented include LdMNPV-BNP, LdMNPV NC_001973 and LyxyMNPV.

Fig. 8. Maximum likelihood molecular phylogenetic analysis of 7 baculoviruses infecting moths of subfamily Lymantriinae. The nucleotide sequences of polh, lef8 and lef9 genes were analyzed. Accession numbers are shown in brackets. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Fig. 9. Nucleotide alignment of v-ubi genes – one from LdMNPV and two from LdMNPV-BNP. Overlapping fragment of v-ubi-A and v-ubi-B marked in red. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) 64 L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66

3.7. DNA photolyase 4. Discussion

The alphabaculovirus analyzed in this study has a gene encod- Among the different forest insects, the gypsy moth is a species ing DNA photolyase (orf34) that is absent in the majority of that is highly susceptible to viral infections. In Poland, viral disease baculoviruses. The photolyases family encompasses enzymes that caused by the gypsy moth was firstly described in 1925 (Nunberg, repair DNA damaged during exposure to the UV light and has 1925) and information on the development and the role of viral functional representatives in many species ranging from disease in reducing population densities of the gypsy moth was Prokaryotes to Eukaryotes (Sancar, 1994; Xu et al., 2008). They provided. In the 1970s, studies on pathogenic microorganisms that are responsible for fixing cyclobutane pyrimidine dimers (CPDs) infect gypsy moth caterpillars in the Biebrzanski National Park created in DNA by UV light. They have been divided into two were conducted (Glowacka-Pilot, 1982). It was concluded that classes on the basis of amino acid sequence divergence early in the nucleopolyhedrovirus was the main factor in natural mortality the evolution process (van Oers et al., 2008). Class II photolyases of L. dispar populations during outbreaks. Although viral diseases are 450–550 amino acids long and have been discovered in viruses caused by gypsy moths occurring in the Biebrzanski National Park and parasites. LdMNPV-BNP orf34 encodes a 493-amino-acid long have frequently been described (Skrzecz, 2000; Sliwa, 1978; DNA photolyase that belongs to class II due to the presence of a Sukovata, 1999), there is no information about the biological flavin adenine dinucleotide (FAD) binding domain (Herniou et al., activity of the virus that infects caterpillars in this area. Thus, we 2011). No transmembrane domains or signal peptides were have presented the characteristics (morphology, infectivity and detected. LdMNPV-BNP DNA photolyase gene is located between genome analysis) of LdMNPV-BNP isolated from L. dispar that v-ubiquitin-B and pp31 and is under control of an early promoter. develops on deciduous trees in the Biebrzanski National Park. This region is also rich in repeated genome elements which Electron microscopy studies confirm that LdMNPV-BNP pro- could contribute to gene acquisition from an unknown source duces occlusion bodies typical of baculoviruses (Hunter-Fujita (baculovirus or insect). The molecular phylogenetic analysis of et al., 1998). The diameter of the OBs, estimated from cross- photolyases amino acid sequences from multiple viruses and silk- sections, was approximately 2 lm. Similar results were obtained worm Bombyx mori is presented in Fig. 10. LdMNPV-BNP does not previously with LdMNPV isolates from France, North America, cluster specifically with any known baculovirus that also possesses Korea, and Turkey (Narang et al., 2001; Yaman et al., 2012) with photolyase. The closest related sequence to LdMNPV-BNP pho- estimated average diameters ranged from 1.5 lm (Korean isolate) tolyase belongs to the group II alphabaculovirus – Sucra jujuba to 2.1 lm (French isolate). Larger diameters of LdMNPV OBs nucleopolyhedrovirus (SujuNPV). In general, the presented results (1–10 lm) isolated from LdMNPV were described in northeastern support previously reported findings that alphabaculovirus regions of the USA (Mazzone and McCarthy, 1981). photolyases have an unknown common ancestor that seems to be In the presented study, the mortality of infected L. dispar L2 different from that of the photolyases found in entomopoxviruses caterpillars reached up from 42 to 100% of insects. The LC50 of 4 (Xu et al., 2008). LdMNPV-BNP was 7 10 OBs/ml, and the LT50 was 9–16 days.

Fig. 10. Maximum likelihood molecular phylogenetic analysis of DNA photolyase amino acid sequences from baculoviruses, entomopoxviruses and silk worm Bombyx mori. As the outgroup DNA photolyase from Rabbit fibroma virus was chosen. LdMNPV-BNP (marked in red) do not specifically cluster together with any baculovirus but DNA photolyase clearly belongs to same baculovirus origin protein family. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) L. Rabalski et al. / Journal of Invertebrate Pathology 139 (2016) 56–66 65

Higher activity of LdMNPV was described earlier only for North Funding information American strains tested on L2 instar larvae (Shapiro et al., 1984). Genome organization and G + C content indicate that LdMNPV- This work was supported by financial grant UMO-2011/03/N/ BNP may be recognized as a different species isolated from NZ9/02215 from National Science Centre (Poland) and 36/UD/ L. dispar, which meets all of the criteria for the classification as SKILLS/2015 from Foundation for Polish Science. Open access alphabaculovirus group II. Because the nucleotide sequences for publication cost supported by the European Commission from the polh, lef-8 and lef-9 genes are very similar to those found in the FP7 project MOBI4Health. LymoNPV in the GenBank database, one can draw the conclusion that these two viruses have a very recent common ancestor or are isolates of the same species. Previously, a number of reports Appendix A. Supplementary material described the possibility of adaptation of a baculovirus to a new host, which was demonstrated by the isolation of a single species Supplementary data associated with this article can be found, in of baculovirus from two or more species of insects (Jehle et al., the online version, at http://dx.doi.org/10.1016/j.jip.2016.07.011. 2006). Moreover, in studies on pathogens of forest pests conducted over a few years at the Forestry Research Institute in Poland, References mortality was not observed when L. dispar caterpillars were exper- imentally infected with polyhedra isolated from environmentally Abbott, W.S., 1987. A method of computing the effectiveness of an insecticide. J. Am. sampled dead L. monacha caterpillars collected in Poland (data Mosq. Control Assoc. 3, 302–303, 1925. not published). Previously published results are inconclusive in Ahrens, C.H., Russell, R.L.Q., Funk, C.J., Evans, J.T., Harwood, S.H., Rohrmann, G.F., 1997. 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