1 Introduction

The major approach for controlling insect pests on different crops all over the world is mainly through chemical or synthetic pesticides. These offer an effective and rapid control of insect pests in major crops. However, excessive and inappropriate use of agrochemicals has resulted in negative ecological and environmental consequences [1]. Reliance on chemical pesticides to manage pest problems in agriculture has aggravated environmental concerns and caused serious health hazards to agricultural workers and rural population [2]. Pesticide residues also raise food safety concerns among domestic consumers and pose trade impediments for export of agricultural commodities [1]. It is in this context that bio-agents, such as bio-fertilizers and biopesticides, have recently become the focus of research and resources in many countries.

Biological pesticides are made from natural sources such as animals, plants, and microorganisms and could be broadly grouped as natural ingredient pesticides, microbial origin pesticides and biochemical pesticides [3]. In general, biological pesticides are safer and less toxic to humans and animals than synthetic pesticides. Moreover, biological pesticides do not cause hazards to nontarget organisms including parasitoids, predators and many vertebrates which make them safer to the habitat and environment. Biopesticides are usually applied to manage rather than to eradicate pests, often incorporating an inbuilt delay factor. Host-specific selective action and safety to nontarget organisms are the hallmark traits of biopesticides. Many species of insect pathogenic microorganisms (bacteria, fungi, viruses, protozoa and nematodes) have been exploited as biopesticides, and some species have been developed into commercial formulations that are being used in many countries [4].

Baculoviruses (BVs) are insect pathogenic double-stranded DNA viruses known for their potential as biological pesticides. More than 700 baculoviruses have been isolated, mostly from larval stages of lepidopteran moths and butterflies [5, 6]. Species in the family Baculoviridae are highly specific to insect hosts from which they have been isolated with few exceptions [7]. BVs are mainly divided into alphabaculoviruses known as nucleopolyhedroviruses (NPVs) and betabaculoviruses known as granuloviruses (GVs). NPVs and GVs differ in occlusion body morphology [8], but both have rod-shaped enveloped nucleocapsids, which are embedded in a protein matrix of the occlusion body that protects the virus from inactivation outside the host after release into the environment [9, 10]. Occlusion-derived virus present in a protein matrix (polyhedrin or granulin) is responsible for the primary infection of the host, while the budded virus is released from the infected host cells later during the secondary infection. This structural protein for GVs is encoded by a granulin gene which is highly conserved among GVs [11] and hence is a popular candidate gene in baculovirus homology studies [12]. A granulovirus infecting semilooper, Achaea janata (order: Lepidoptera; family: Noctuidae), an insect pest on castor beans (Ricinus communis L.), was first isolated by Vimala Devi [13], restriction endonuclease analysis of its genome was carried out [14], symptoms of infection were studied [15], an aqueous formulation was developed [16], and its bio-efficacy and mammalian safety were tested [17]. In this study, we identified the granulin gene sequence and characterized the protein from the Hyderabad isolate of Achaea janata granulovirus (AcjaGV). We compared the granulin nucleotide sequence and its deduced amino acid sequence with several known GVs. Established the phylogenetic relationship of AcjaGV with GVs infecting insect hosts from the moth family of Noctuidae in Lepidoptera, which are among the most serious pests infesting several commercial crops.

2 Materials and methods

2.1 Virus Propagation, Extraction of Viral Genomic DNA and PCR

AcjaGV was propagated in vivo by administering a dose of 5 × 104 OB/ml to healthy late third instar larvae. The infected larvae were collected in single distilled water and frozen for further use. Thawed samples were homogenized and subjected to differential centrifugation initially, at low speed (400 g for 5 min), followed by high speed (11000 g for 30 min) to pellet the virus and suspended in sterile distilled water [17]. To extract virus DNA, 0.01 M EDTA and proteinase-K were added to purified occlusion bodies (OBs) to a final concentration 2 mg/ml and the mixture was incubated at 37 °C for 90 min. Sodium carbonate solution (Na2CO3) was added to the above mixture to final concentration 0.1 M and incubated at 37 °C for 30 min, and further, the viral genomic DNA was extracted through the QIAamp DNA Mini kit (Qiagen).

Polymerase chain reaction (PCR) was carried out in a thermal cycler (Veriti; ABI-Applied Biosystems) with the following cycling parameters: 94 °C for 2 min as initial denaturation, followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and 72 °C for 20 min as final extension by using degenerate primers (Gra-F 5′ ATG GGA TAY AAC ARA KCW YTR MGK TAY AGY MRH CAC 3′ and Gra-R 5′ TTA RTA VGC BGG DCC DGT RWA YAR WGG YAC RTC 3′) specific to granulin gene, designed based on the nucleotide sequence of other granulovirus granulin genes using multiple sequence alignment (CulstalW). PCR was performed in a 20 µl reaction volume containing 20 pmol of each primer, 10 × PCR buffer, 1.4 mM MgCl2, 0.1 mM dNTPs, 25 ng of AcjaGV genomic DNA and 1 U of Taq DNA polymerase (Qiagen). The amplified products were resolved in 1.0 % agarose gel, stained with ethidium bromide (10 µg/ml), and visualized in a gel documentation system.

2.2 Molecular Cloning and Sequencing

The PCR amplified fragment was eluted using QIAquick gel extraction kit and ligated into the general purpose-cloning vector, InsT/Aclone, and transformed using Escherichia coli (DH5∞) cells. White colonies (colonies harboring the insert) were maintained on Luria–Bertani agar medium (LBA) containing ampicillin (100 mg/ml), and further it was incubated overnight at 37 °C and stored at 4 °C until further use. Plasmids were isolated using SureSpin Plasmid Mini kit (Genetix). Sequencing was carried out in an automated sequencer ABI DNA analyzer Model 3730xl (Applied Biosystems, USA) using M13 universal primers, both in forward and in reverse directions.

2.3 Phylogenetic Analysis of AcjaGV Granulin

Granulin gene nucleotide sequence and deduced amino acid sequences of 17 GVs and polyhedrin gene sequences of 13 NPVs were compared individually to analyze the extent of homology with AcjaGV granulin gene of interest. The evolutionary history was inferred using the neighbor-joining method [18]. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed [19]. The evolutionary distances were computed using the Poisson correction method [20] in the units of number of base substitutions per site. Phylogenetic analysis was done using MEGA4 software [21].

2.4 Extraction of AcjaGV Granulin Protein and their SDS-PAGE and 2D-Gel Electrophoresis

Purified virus capsules were heat treated at 70 °C for 60 min, and then phenylmethylsulfonyl fluoride was added to make up the final concentration to 300 mM. These solutions were incubated in equal volume of 0.1 M sodium carbonate at 37 °C for 30 min and centrifuged at 7000 rpm for 20 min to remove undissolved virus capsules. Further, the granulin was precipitated by adding 5 vol of cold acetone and the precipitate was pelleted by centrifugation at 10,000 g for 15 min. The granulin pellet was resuspended in distilled water [14]. Extracted granulin protein from AcjaGV was analyzed for heat shock proteins by SDS-PAGE with 1.5-mm-thick resolving gels of 12 % acrylamide overlaid with 5 % stacking gels [22]. The protein profile of isolates at ambient and elevated temperature was compared.

The protein concentration was determined by Lowry protein assay. Isoelectric focusing electrophoresis was carried out with 7 cm (pI 3–10) IPG strips, at 20 °C, according to the manufacturer’s instructions (Bio-Rad). Briefly, the strips were rehydrated with 50 V for 13 h.

2.5 In-Gel Digestion

Gel pieces were extracted manually using a handheld pipette with a trimmed polypropylene tip. Destaining of the gel pieces and reduction/alkylation/digestion of the contained proteins was done by adapting published procedures [23]. Destaining and cleanup were accomplished by incubating the gels in water, ammonium bicarbonate, 50 % acetonitrile and acetonitrile. After reduction with dithiothreitol and derivatization with iodoacetamide, the gel pieces were infused with freshly prepared trypsin (30 ml) and incubated overnight at 37 °C.

2.6 Extraction of Peptides

The digest solution was removed and transferred into a fresh microcentrifuge tube; 150 µl of extraction buffer (0.1 % TFA, 50 % ACN) was added to cover the gel pieces and kept in a shaker for 20 min. The extraction buffer was removed, and step 2 was repeated thrice. After the third extraction, the tube was allowed for drying in SpeedVac. Ten microliters of TA buffer was added and suspended dried spots by vortexing and short spun and spotted it on the MALDI (matrix-assisted laser desorption/ionization) plate after thorough mixing with matrix (Sinapinic acid) at 1:1 concentration.

2.7 Protein Identification

Spots were identified using peptide mass fingerprinting (PMF) and MS/MS (mass spectrometry/mass spectrometry) of the ten most intense ions acquired using automated data acquisition. Spectra were batch analyzed using mass spectrometer (Applied Biosystems). The mass list for each sample was analyzed using the program MASCOT, Matrix Science search engine accessed (http://www.matrixscience.com) on March 10, 2012, and rapid protein identification software (MASCOT) using mass spectroscopy data assuming one missed cleavage, carboxymethylation and methionine oxidation was used as modifications.

The lists of masses were compared against the Mascot Search Data Base (MSDB) and virus protein database.

2.8 Restriction Enzyme Digestions

The prime objective of our study was to identify AcjaGV strain from other granuloviruses through PCR–RFLP marker analysis based on the unique restriction sites in the granulin gene. Thus, the restriction sites of all the 18 retrieved granulin gene of various granuloviruses were mapped using pDRAW32 (http://www.acaclone.com) [24].

2.9 Virtual Restriction Fragment Length Polymorphism (RFLP) Patterns

RFLP patterns of all the 18 granuloviruses were studied through pDRAW32 taking Promega 1-kb DNA ladder. Virtual RFLP patterns from in silico digestions of granulin gene of 18 granuloviruses mapped by 12 restriction enzymes were used for simulated digestions: BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, MluI, PstI, PvuII, SalI, SmaI and XhoI.

3 Results and Discussion

Nucleotide and predicted amino acid sequences of AcjaGV granulin gene are shown in Fig. 1. Granulin gene ORF comprised of 753 nucleotides encoding a peptide of 250 amino acids, which has similarity with granulin protein from other GVs (Table 1).

Fig. 1
figure 1

Nucleotide and predicted amino acid sequences of AcjaGV granulin gene (matrix protein in OBs). The predicted amino acid is represented by the one letter code designation below the nucleotide sequence. * Indicates termination codon

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Table 1 Comparison of granulin gene and protein characteristics across GVs
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Cladograms based on granulin gene and amino acid sequences are shown in Fig. 2. In both cases, the phylogenetic tree showed two branches: one representing the GVs and the other NPVs. AcjaGV was closest to Xestia c-nigrum granulovirus (XecnGV), Helicoverpa armigera granulovirus (HearGV), Pseudaletia unipuncta granulovirus (PsunGV), Trichoplusia ni granulovirus (TrniGV) and Spodoptera litura granulovirus (SpliGV). There were also some distances observed phylogenetically in spite of this close association, all of which infect insects belonging to the family Noctuidae suggesting that these GVs share a common ancestor and were separate from the rest (Fig. 2).

Fig. 2
figure 2

Cladogram showing phylogenetic relationship of AcjaGV (*) with other GVs based on nucleotide (a) and amino acid sequences (b) of granulin gene. Tree generated using MEGA4 software (v4.0.2) by neighbor-joining method (1000 boot straps). GenBank accession numbers are also shown

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Translated amino acid sequence of AcjaGV granulin gene using BLASTp search showed highest homology with granulin proteins as expected (Table 2). The degree of identity ranged between 73.7 and 84.7 % with granulins of other GVs. The closest homology was observed among the granulins of the same cluster comprising of SpliGV (84.7 %), HearGV (84.3 %), PsunGV (84.3 %), XecnGV (84.3 %) and TrniGV (83.9 %). Lowest homology was observed with Pieris brassicae GV (PibrGV) with an identity of 73.7 %. The degree of identity of AcjaGV granulin with the polyhedrin protein of Autographa californica nucleopolyhedrosis viruses (AucaNPV) was low at 52 %.

Table 2 Percent identity of amino acid sequences of the AcjaGV granulin with other known GVs and AucaNPV sequence
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Multiple alignment of the deduced amino acid sequence of AcjaGV granulin with known GVs and polyhedrin from AucaNPV is shown in Fig. 3. Highly conserved regions were noticed at positions 58–83, 86–139 and 206–245 near C terminus (Fig. 3). The region of granulin is highly conserved among GVs [25, 26]. The non-conserved regions were found in the N terminus between amino acid residues 1–4 and 20–55. Among the granulin amino acid residues, arginine, cysteine and proline were highly conserved among GVs. However, these three residues were less conserved when compared with AucaNPV polyhedrin at corresponding positions of 10, 83, 169, 207 and 211 (Fig. 3). Highly conserved regions play a major role in the secondary and tertiary structure of granulin and polyhedrin proteins [27]. Apparently, the shape of OBs is determined by several factors including the protein sequence of granulin or polyhedrin and interactions with other proteins on the virion surface during occlusion [28].

Fig. 3
figure 3

Multiple alignment of AcjaGV granulin amino acid sequence with other known GVs granulin and AucaNPV polyhedrin amino acid sequences. Dots indicate homology of other GVs and AucaNPV sequences with AcjaGV. * Indicates conserved amino acid residues

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Polyhedrin protein of AucaNPV contains a nuclear localization signal (KRKK). The amino acid sequence in AcjaGV granulin at the corresponding position was RRKR which appears to be unique among the known GVs. Variations found at corresponding positions for other GVs were the amino acid sequence RHKE (HearGV, XecnGV, TrniGV and PsunGV), RRKK (Adoxophes orana GV (AdorGV), Cryptophlebia leucotreta GV (CrleGV), Cydia pomonella GV (CypoGV), Epinotia aporema GV (EpapGV), Harrisina brillians GV (HabrGV), Phthorimaea operculella GV (PhopGV), Plutella xylostella (PlxyGV) and SpliGV), RHKK (Andraca bipunctata GV (AnbiGV) and Choristoneura fumiferana (ChfuGV)), HKKD (Agrotis segetum GV (AgseGV)) and KHKK (Pieris brassicae (PibrGV) and Pieris rapae (PiraGV)). The lack of a nuclear localization signal in granulin suggests that it is not required for GV occlusion, and this corresponds to the difference in cytopathology of GVs and NPVs. Replication in NPVs leads to the formation of occlusion bodies exclusively localized in the nucleus. During the replication process of GVs, nucleus lyses and the virions get occluded in a mixture of nucleoplasm and cytoplasm [29]. Cells infected with a recombinant AucaNPV in which polyhedrin gene was replaced with TrniGV granulin gene produced large cuboidal inclusions in both the cytoplasm and the nucleus [30]. However, replacement of RHKE in TrniGV granulin gene with the KRKK nuclear localization signal resulted in an increased localization of this protein in the nucleus [30]. This perhaps suggests that the primary structure of granulin is not solely responsible for ovoid-shaped occlusions, but other cellular and viral factors contribute to its occlusion morphology.

The separation of protein by SDS-PAGE showed that AcjaGV granulin, one of the major OB matrix proteins, banded at mean of 29.5 ± 0.7 kDa granulin (Fig. 4), which agreed well with the size reported earlier by Singaravelu & Ramakrishnan [14]. Further, mass spectroscopy of peptide fractions followed by Mascot search (Fig. 5) established the identity of the granulin protein of AcjaGV which had a similarity score of 87 with peptide fractions of SpliGV granulin protein, which belongs to the Noctuidae family of Lepidoptera (Table 3). Similar kind of mass spectroscopy analyses could identify the polyhedrin protein from Bombyx mori NPV (BomoNPV) and AucaNPV [31].

Fig. 4
figure 4

Molecular weight of major OB matrix protein (granulin) in 12 % SDS-PAGE. Lane M is a broad range protein marker, and lane 1 is AcjaGV granulin protein (arrow)

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Fig. 5
figure 5

Mass spectrum with Mascot analysis (mass: 29,365; score: 87). Protein score is −10*Log (P), where P is probability that the observed match is a random event. Protein scores greater than 38 are significant (P < 0.5)

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Table 3 Proteins identified by MALDI-TOF/MS from Achaea janata GV
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Betabaculoviruses are categorized into different types based on tissue tropism during the in vivo infection process. After ingestion by the larval instars of insect host, type I GVs infect the midgut epithelium and subsequently infect fat body tissue. Phylogenetically related type I GVs are expected to exhibit a similar infection pathway. Nucleotide and deduced amino acid sequences of SpliGV granulin gene showed close homology with other type I GVs like TrniGV and XecnGV [32]. Type I GVs reportedly have a slow speed of kill compared to type II GVs as they require several 100-fold higher median lethal dose (LD50) and twofold to threefold slower median lethal time (LT50) as observed in the cases of SpliGV [33], XecnGV, PsunGV [34], PlxyGV [35] and AcjaGV [17]. Type II GVs like CypoGV are highly infectious to their hosts with an LD50 of 1–5 occlusion bodies larva−1 [36]. The difference in speed of action of GVs may lie in the range of larval tissues infected by a virus with higher virulence associated with a broader range of tissues infected as in the case of type II GVs [37]. However, differences in slow and fast acting GVs cannot be attributed to tissue tropism alone but could lie in their genetic differences [38]. Our finding that AcjaGV granulin gene and amino acid sequences share significant homology with SpliGV, TrniGV and XecnGV, and all type I GVs with a similar mode of action reinforced that the highly conserved granulin gene is a good candidate for phylogenetic studies of GVs.

Virtual restriction digestion of granulin gene of different GVs revealed a unique restriction pattern for the test AcjaGV strain with BamHI, EcoRI, HindIII, KpnI, PstI, EcoRV and PvuII. In case of granulin of other GVs, restriction sites for these enzymes were not unique. All the granulin genes examined did not posses sites for XhoI and SmaI (Table 4). More restriction sites in granulin gene of the 18 GVs subjected to in silico analysis 11 GVs revealed a common restriction site at 180 bp with BamHI. Similarly, eight GVs had a common EcoRI restriction site at 703 bp, while four GVs have common HindIII restriction site at 259 bp and five GVs EcoRV restriction site at 153 bp (Fig. 6; Table 4). AcjaGV granulin gene was unique with SalI two restriction sites at 35 bp and 61 bp, which were absent in other 17 GVs (Fig. 7; Table 4).

Table 4 Restriction sites present in granulin genes of AcjaGV and other granuloviruses with different restriction enzymes using pDRAW32 software
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Fig. 6
figure 6

In silico EcoRI, MluI and SalI restriction site locations in AcjaGV granulin gene sequence (pDRAW32 software)

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Fig. 7
figure 7

Virtual RFLP patterns from in silico digestions of granulin gene fragments from 18 granuloviruses. Recognition sites for the following 12 restriction enzymes are used in the simulated digestions: BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, MluI, PstI, PvuII, SalI, SmaI and XhoI. MW: Promega 1-kb DNA ladder

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In order to distinguish the AcjaGV strain with other GVs, a PCR–RFLP marker was identified based on restriction sites present in the amplified portion of the granulin gene. Analysis of REN sites in published granulin sequences showed one restriction site for EcoRI at nucleotide position 703 in GVs, viz. XecnGV, HearGV, TrniGV, PsunGV and PlxyGV. The EcoRI site was at nucleotide position 286 in CypoGV, ChfuGV, CrleGV and PtopGV. No EcoRI restriction site was found in granulin gene sequence of SpliGV, AnbiGV, AgseGV and HabrGV. In AcjaGV granulin gene sequence, EcoRI restriction site was located at 331 bp (Fig. 6) which was unique to AcjaGV. The EcoRI digest of granulin PCR amplified gene resulted in two fragments sized 331 and 442 bp in 2 % agarose gel (Fig. 8) which corresponded well with the in silico restriction site location shown in the granulin gene sequence using pDRAW32 software for the PCR product (Fig. 6).

Fig. 8
figure 8

Granulin gene is digested with EcoRI (lane 1) and MluI (lane 2) showing two bands in 2 %agarose gel. Marker (100-bp ladder)

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MluI restriction digestion pattern of AcjaGV earmarked a restriction site at 542 bp, whereas in other five GVs this site was not observed at same position. Analysis of REN sites in published granulin sequences showed restriction site for MluI in SpliGV, AnbiGV, EpapGV, CypoGV and PiraGV at nucleotide positions 428, 425, 307, 237 and 237, respectively (Table 4).

In order to confirm the virtual restriction enzyme digestion pattern of AcjaGV, the in vivo experiment was executed. A similar digestion pattern of the gene was visualized in silico analysis. The in silico fragmentation pattern observed with AcjaGV was further confirmed by subjecting the southern blotting and PCR–RFLP. The southern hybridization with a homology a homologous probe (AcjaGV granulin PCR product) led to the identification of REN fragments containing AcjaGV granulin gene. It was revealed that EcoRI and MluI restriction enzymes produced two hybridization bands because of the internal restriction site present in the granulin gene (Fig. 9). In case of SalI restriction enzymes, three bands were observed in in silico analysis due to two internal restriction sites present in the granulin gene at 35th and 61st positions. However, in vivo only one hybridization band was observed because of the low molecular weight of two bands (35 and 26 bp) which were not observed in southern hybridization. No clear hybridization band was seen in SalI restriction pattern in southern hybridization.

Fig. 9
figure 9

Southern hybridization with homologous granulin gene probe to REN fragments of AcjaGV DNA, lane L is 1-kb plus ladder included as a molecular size marker

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4 Conclusion

In this paper, we established the sequence identity of granulin gene of the lesser known AcjaGV and characterized its protein product. We identified the phylogenetic relationship with other known GVs based on granulin gene nucleotide and deduced amino acid sequences, and also the similarity of peptide fractions of its protein. Interestingly, AcjaGV was closely related to GVs infecting larvae of the Noctuidae family of lepidopteran insects, all of which apparently belong to type I GVs that share a similar tissue tropism and speed of kill of their larval hosts. However, the degree of genetic relatedness of AcjaGV with other type I GVs and its specific differences with fast acting type II GVs is to be probed further. The presence of unique restriction sites in AcjaGV gene for three different restriction enzymes, which were not seen in other GVs, makes this tool a cost-effective and rapid method for identification of GVs from different sources. The in silico virtual digestion pattern of AcjaGV was further supported by the evidence of PCR amplification and southern hybridization techniques. The utilization of such tools can help in early detection of viruses of Lepidoptera order.