2.1 Isolation of NPV occlusion bodies and data analyses

The collected larvae were brought to the laboratory and stored at −20 °C and then evaluation was carried out to find percent infected larvae from collected larvae. The infected larvae were dissected and wet smears were prepared to examine under phase contrast microscope for the presence of occlusion bodies (OBs). The occlusion bodies (OBs) were isolated by following the standard methodology (Hussain et al., 2019) with few modifications (Additional details in Supplementary file 3).

2.2 Virus DNA extraction

The total DNA was extracted from larvae exhibiting symptoms of NPV disease using the cetyltrimethylammonium bromide (CTAB) method, following the protocol (Sivakumar et al., 2020). Subsequently, all DNA extracts underwent a 1:100 dilution in sterile distilled water (SDW) before being utilized in RCA amplifications (Pavan et al., 2024). Quantification and adjustment of the isolated DNA concentration to 0.1 g/l were performed using a NanoDropTM 1000 Spectrophotometer from ThermoScientific, USA. Verification of the size and purity of the DNA was achieved by loading 1 µl of the sample DNA and 1 µl of control genomic DNA onto a 0.6 % agarose/EtBr gel, along with a 1 kb Plus DNA ladder. This gel electrophoresis confirmed the size and purity of the extracted DNA (Additional details in Supplementary file 3).

2.3 PCR amplification of polyhedrin gene of SpfrNPV strain AAUBC1 and AP2

In this study, the conserved polyhedrin gene (polh) of NPV was targeted using species-specific primers designed from the NCBI database. The PCR reaction, following Thermo Scientific's protocol, employed 35 cycles to amplify the marker (Sivakumar et al., 2020). The amplified products were visualized on a 1.2 % ethidium bromide-stained agarose gel, alongside Gene Ruler 100 bp DNA ladder, confirmed fragment sizes. Sequencing at Barcode Bioscience, Bengaluru, India, facilitated SpfrNPV identity confirmation. BLAST analysis against NCBI GenBank verified nucleotide sequence homology. The sequences were deposited in GenBank with accession numbers PP025827 and PP025828 for the isolated SpfrNPV strains AAUBC1 and AP2 (Additional details in Supplementary file 3).

2.4 Gene structure prediction

The gene structure, including exons and introns, was determined by comparing of open reading frames (ORFs) and the SpfrNPV polyhedrin genes (Guo et al. 2007). As described, the structural details were visualized using GSDS 2.0 (https://gsds.cbi.pku.edu.cn) Fig. 1.

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Fig. 1

Gene structure schematic diagram for SpfrNPV AAUBC1 and AP2 polyhedrin genes. Exons were demonstrated by filled green boxes and untranslated region(UTR) was displayed red boxes, and introns represented by dark blue lines.

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2.5 Phylogenetic analysis

A total of 102 polyhedrin sequences were utilized to construct the phylogenetic tree. The sequences were aligned using the Clustal W program, employing default gap penalty parameters with a gap opening of 10 and an extension of 0.2. Subsequently, a neighbor-joining tree was constructed using MEGA 6.0, adopting a p-distance model and implementing pairwise deletion of gaps (Tamura et al., 2013). To assess the robustness of the tree branches, bootstrap support was determined by resampling amino acid positions 1000 times. Additionally, conserved domains within the identified SpfrNPV strain AAUBC1 and AP2 were predicted using the SMART tool (https://smart.embl-heidelberg.de/), as outlined by Letunic et al. (2014) (Supplementary file 1; Fig.S3 & Fig. 2).

2.6 Motif analysis

Following the BLAST results, the protein identified as a key player in disease incidence, specifically the symptom-determinant protein, was subjected to motif discovery and pattern analysis. The MEME software, version 4.12.0, available on the online server (https://meme-suite.org/index.html), was employed for this purpose. In this analysis, the parameters used for motif discovery were set as follows: minimum width = 6, maximum width = 10, and the maximum number of motifs to find = 6. These parameters guided the MEME software in identifying and analyzing motifs within the protein sequence, providing valuable insights into potential functional patterns associated with the disease-related protein.

2.7 Predicting protein structures for SpfrNPV AAUBC1 and AP2 polyhedrin from S. frugiperda

For the prediction of the structure of the identified SpfrNPV AAUBC1 and AP2 polyhedrin, we employed Phyre2 (Protein Homology/Analogy Recognition Engine V 2.0), following the methodology (Rudra Gouda et al., 2023). The prediction process was conducted using the online tool available at https://www.sbg.bio.ic.ac.uk/∼phyre2/html/page.cgi?id=index. The prediction involved the utilization of intensive mode and ab initio techniques to achieve comprehensive protein modeling and to explore potential functional roles. Batch processing in expert mode allowed for simultaneous sequence processing, enhancing efficiency. While alternatives such as the Swiss model and I-Taser were considered, Phyre2 was chosen for its superior modeling capabilities. The quality of the predicted models was assessed using various tools, including SAVES v5.0 (https://servicesn.mbi.ucla.edu/SAVES), ProSA, and Qmean. These assessments provided valuable insights into the reliability and accuracy of the predicted protein structures.

2.8 In-silico docking

2.9 Bioassay

The determination of the median lethal concentration (LC₅₀) of the isolated strain of SpfrNPV AAUBC1 on 2nd (3–5 days old), 3rd (6–7 days old), and 4th (6–7 days old) instar larvae was carried out through a leaf disc bioassay method (Magholi et al., 2014) with certain modifications (Additional details in Supplementary file 3). The mortality rates of the larvae were documented every 24 h for a duration of up to 9 days post-inoculation Median lethal concentrations (LC₅₀) were determined by probit analysis using SPSS software. The mortality rate of targeted insects was corrected using Abbott’s formula (Abbott, 1925). SPSS 21.0 software was used for all the analyses.

3.1 Stage-wise NPV infection of fall armyworm in maize of Panchmahal and Mahisagar district during kharif, 2021

An extensive study in the Panchmahal and Mahisagar districts assessed the prevalence of nucleopolyhedrovirus (NPV) infection in S. frugiperda larvae during maize crop development (Supplementary file 1; Table S2 & Table 2). NPV prevalence peaked during cob formation (28.74 %), followed by flowering and tasseling (20.23 %), and the vegetative stage (13.93 %) (Table 1). Block-specific analyses revealed variations, with Jambughoda having the highest vegetative stage infection (20.41 %) and Khanpur the lowest (9.09 %). Ghoghamba showed the highest NPV infection during flowering and tasseling (27.47 %), while Santrampur had the lowest (15.79 %). Godhra had the highest infection during cob formation (34.44 %), and Morwa had the lowest (24.66 %). Correlation analysis found significant positive relationships between NPV infection and different crop stages (Table 2). Regression equations quantified these relationships (Table 2). Month-wise mean data analysis elucidated the relationship between weather conditions and NPV infection, with the highest infection occurring in September (28.74 %), followed by August (20.23 %) and July (13.93 %) (Supplementary file 1; Table S3).

3.2 Correlation between weather parameters and NPV infection in larvae of fall armyworm, S. frugiperda, infesting maize

The detailed analysis of weather parameters during the kharif season in 2021 sheds light on the correlation between environmental conditions and nucleopolyhedrovirus (NPV) infection in fall armyworm (S. frugiperda) larvae (Table 3). Maximum temperature displayed a non-significant but negative correlation (r = −0.959), suggesting that elevated temperatures are associated with decreased natural NPV infection. In contrast, minimum temperature exhibited a negative and significant correlation (r = −0.998*), indicating an inverse relationship with lower temperatures and enhanced NPV infection rates. Morning relative humidity showed a highly significant positive association (r = 0.999**), emphasizing the crucial role of high morning humidity in facilitating increased NPV infection rates. Evening relative humidity exhibited a positive association (r = 0.899), though non-significant, suggesting that higher evening humidity is linked to elevated NPV infection. Conversely, a negative but non-significant correlation between sunshine hours and NPV infection implies that increased sunlight exposure may be associated with decreased natural NPV infection. The analysis of rainfall indicated a non-significant negative correlation (r = −0.768), suggesting that higher rainfall is not strongly associated with enhanced NPV infection in fall armyworm larvae. Regression equations for temperature, humidity, and sunshine hours provide quantitative relationships (Table 3). For example, the equation Y = 153.26 + (−4.044) × (R² = 0.920) for maximum temperature indicates a corresponding decrease in natural NPV infection for every unit increase in temperature. Similarly, equations for morning and evening relative humidity express an increase in NPV infection for every unit rise in humidity. The equation Y = 40.86 + (−6.77) × (R² = 0.99) for sunshine hours conveys a decrease in natural NPV infection with every unit increase in sunlight exposure.

3.3 Prediction of domains and their function in SpfrNPV strain AAUBC1 and AP2 in S. frugiperda

Utilizing SMART bioinformatics and subsequently validating through the conserved domain search tool of NCBI, conserved domains in the polyhedrin of SpfrNPV strain AAUBC1 and AP2 from S. frugiperda were predicted. The analysis, conducted on the amino acid sequences of the coding region (CDS), revealed the presence of two functional domains in both polyhedrins. The predominant domains identified in SpfrNPV AAUBC1 and AP2 polyhedrins were associated with the polyhedrin pfam family. This suggests a conserved structural element within the polyhedrin protein. The inference drawn from this observation is that the polyhedrin family serves a Gene Ontology (GO) function, specifically contributing to structural molecule activity. This functional annotation was corroborated by SMART Bioinformatics (Letunic et al., 2014), supporting the structural role of the polyhedrin family. To visually represent these domains and their functions, the Quick GO EMBL tool was employed, and the annotated domains are illustrated (Supplementary file 1; Fig.S3). This figure comprehensively depicts the predicted domains and their attributed functions within the context of structural molecule activity. These findings contribute to understanding the functional aspects of polyhedrin in the context of S. frugiperda Nucleopolyhedrovirus.

3.4 Gene structure of SpfrNPV AAUBC1 and AP2 polyhedrin genes

The gene structure prediction for the SpfrNPV AAUBC1 and AP2 polyhedrin genes was accomplished using GSDS 2.0 software. The intron structure was deduced in this process, and exon boundaries containing functional domains (polyhedrin) were identified in the NPV polyhedrin gene. The genomic organization of polyhedrin genes was elucidated through this analysis. Remarkably, the majority of polyhedrin genes are covered by functional exons. A Single functional exon was found in SpfrNPV AP2 polyhedrin. However, SpfrNPV AAUBC1 polyhedrin has two exons separated by an intron, constituting a non-coding region. This structural feature, commonly referred to as interrupted genes, is highlighted (Fig. 1). Investigating the exon–intron arrangement within the polyhedrin gene of NPV provides a promising avenue for gaining new insights into the evolutionary trajectory of this gene family.

3.5 Phylogenetic analysis of polyhedrin of SpfrNPV strain AAUBC1 and AP2

A neighbour-joining tree was constructed to elucidate the evolutionary relationships among polyhedrin sequences, incorporating sequences from two SpfrNPV strains, AAUBC1 and AP2, in addition to 100 sequences from various insect species (Supplementary File S2). The resulting phylogenetic tree demonstrated a distinct clustering, where all polyhedrin sequences from S. frugiperda formed a separate group, indicating a species-specific divergence (Fig. 2). Intriguingly, both SpfrNPV strains, AAUBC1 and AP2, clustered together with polyhedrin sequences from the same S. frugiperda species. This suggests a close evolutionary relationship between the polyhedrins of these two strains, reinforcing their shared genetic lineage. Furthermore, the analysis revealed that polyhedrin sequences from S. frugiperda share a notable evolutionary affinity with polyhedrins from other Spodoptera genus species, such as exigua, litura, eridania, and cosomioides. This observation underscores the genus-specific nature of NPV groups, indicating a distinctive evolutionary history within each genus. Notably, the variation among species within the same genus is evident, highlighting contrasting evolutionary trajectories. In summary, the phylogenetic analysis provides insights into the evolutionary relationships of SpfrNPV polyhedrin, emphasizing both species-specific clustering within S. frugiperda and a broader genus-specific association with polyhedrins from other Spodoptera species. This information contributes to our understanding of the evolutionary dynamics of NPV polyhedrins across different insect species.

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Fig. 2

Phylogenetic analysis of the amino acid sequences of the NPV polyhedrin from different insect species (our accession is indicated in red color) in the context of various NPV Polyhedrin. The NPV polyhedrin was used to create a neighbor-joining tree, which was based on the nucleotide sequences of 102 different NPV isolates. Boots trap values were calculated with 1000 replications.

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3.6 Motif pattern analysis of polyhedrin from SpfrNPV strain AAUBC1 and AP2

Motif analysis was conducted using MEME (version 4.12.0) with specific parameters to discern conserved motifs in the polyhedrin sequences of SpfrNPV strains AAUBC1 and AP2 from S. frugiperda. Employing a minimum width of six, a maximum width of 10, and limiting the identification to a maximum of six motifs ensured the stringent identification of biologically relevant motifs. The results revealed six distinct motifs in both NPV strain AAUBC1 and AP2 polyhedrins, denoted as AQHALRCDPD, MNLHSEYTHS, TWTRFMEDSF, SLAKKGGGCP, PIVNDQEIMD, and MRPTRPNRCF (Fig. 3A). Notably, all six motifs were conserved in both strains, indicating a shared motif pattern. Further analysis demonstrated an identical motif pattern in both strains, characterized by the presence of TWTRFMEDSF, PIVNDQEIMD, MRPTRPNRCF, AQHALRCDPD, SLAKKGGGCP, and MNLHSEYTHS. The statistical significance of this motif pattern was confirmed with p values of 4.61e-60 for NPV strain AAUBC1 and 1.11e-58 for AP2, underscoring the robust conservation of these motifs (Fig. 3B). This consistent motif pattern suggests a functional significance in the polyhedrin protein of SpfrNPV strains AAUBC1 and AP2, possibly contributing to their structural and functional roles in the viral life cycle. The stringent parameters applied in the motif analysis enhance the confidence in the biological relevance of the identified motifs.

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Fig. 3

Motif and protein structural details of polyhedrin of SpfrNPV strain AAUBC and AP2 from S. frugiperda. 3A: Motifs identified in polyhedrin of SpfrNPV strain AAUBC and AP2 from S. frugiperda. Motifs were discovered by using MEME tool. Heights of the symbols within a stack indicate the relative frequency of the amino acids at that position. Parameters used for motif discovery were: minimum width = 6, maximum width = 10, maximum number of motifs to find = 6. 3B: Motif pattern identified in polyhedrin of SpfrNPV strain AAUBC and AP2. Color boxes in figures are motifs and the pattern of that motif is indicated in color coded box at bottom. Both the polyhedrin motif pattern are less than p- value 0.0001 and are statistically significant. Length of the bar of each of polyhedrin indicates length of CDS region used in drawing motifs. The overall height of a stack indicates the degree of sequence conservation at that position. 3C: Ligand (Chitinase in red) interaction with the binding pocket in protein (Polyhedrin of S. frugiperda NPV in blue). (I) Surface structure (II) Stick structure.

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3.7 Deducing the protein structure of SpfrNPV polyhedrin identified in S. frugiperda

Our study successfully employed the Phyre2 tool to predict the theoretical protein structure of S. frugiperda Nucleopolyhedrovirus (SpfrNPV) polyhedrin (Table 4). The SpfrNPV genes deduced structures, AAUBC1 and AP2, exhibited significant similarities, with 89 % and 83 % resemblance, respectively, to the crystal structure of the infectious baculovirus polyhedral protein from Wiseana signata NPV. The identified polyhedrin molecules displayed distinctive structural features, including an extended N-terminal alpha helix projecting from the main part of the molecule. Moreover, a compact β-sandwich domain with three-layered β sheets and peripheral small alpha helices was evident. These characteristics align with the structural findings reported by Mori et al. in 2010. As of now, the NMR/crystal structures of only one polyhedral NPV have been resolved and are accessible in Entrez's 3-D structure database at NCBI. However, the observed similarities with W. signata NPV polyhedral protein suggest a conserved structural pattern among polyhedrins in different NPVs. The limited availability of crystal structures emphasizes the need for further research to unravel the detailed structural features of polyhedrins. A more extensive database of crystal proteins will contribute to a comprehensive understanding of the structural intricacies, facilitating advancements in the study of NPV polyhedrins.

Table 4 Predicted protein structure and function of polyhedrin in SpfrNPV Strain AAUBC1 and AP2 of S. frugiperda.

Gene name	NMR structure	Confidence	Coverage%	Identity%	Descriptor	Functional Keywords	Biological source	Total formula weight	Total number of polymer chains
SpfrNPV AAUBC1 and SpfrNPV AP2		100 %	81 %	89 % (AAUBC1) 83 % (AP2)	Crystal structure of infectious baculovirus polyhedra	jelly-roll, disulfide bond, domain swapping, viral protein	Wiseana signata NPV	28801.88	1

Column 2 indicates the predicted NMR (nuclear magnetic resonance structure) of polyhedrin of SpfrNPV strain AAUBC1 and AP. Column 6 indicates the match of our polyhedrin of SpfrNPV strain AAUBC1 and AP protein structure to the respective NPV proteins of other insects. Column 8 shows the organism from which our polyhedrin structure matches the polyhedrin of that organism.

3.8 Molecular docking studies

The interaction between the polyhedrin of S. frugiperda and a chitinase enzyme was investigated by in silico docking analysis. The binding energy was used as a criterion to evaluate the affinity between the polyhedrin protein and the chitinase enzyme. We have chosen the chitinase enzyme because when insects are infected by NPV, their immune system is activated to defend against the virus. Chitinase production is one aspect of the insect's defense mechanism (Abulikemu et al., 2021). The chitinase enzyme may play a role in breaking down the chitinous components of the virus, such as the proteinaceous matrix or the viral occlusion bodies, which are structures that protect and transmit the virus. The breakdown of chitin by chitinase could potentially hinder the spread and replication of the NPV within the insect host. Additionally, chitinase may be involved in the degradation of infected cells or viral particles, contributing to the insect's defense against the virus (Abulikemu et al., 2021). Our objective was to optimise these interactions to achieve the most favourable binding with the lowest energy requirements. Our results reveal that ligand compound established robust bonds with one or more amino acids within the active pockets of the polyhedrin of S. frugiperda. Furthermore, chitinase enzyme exhibited very high binding energies of 9.4 kcal/mol (Supplementary file 1; Table.S4 & Fig. 3C).

3.9 Bioassay

The bioassay conducted on the isolated strain of SpfrNPV AAUBC1 against 2nd, 3rd and 4th instars larvae of S. frugiperda in laboratory conditions revealed a notable variation in its biological activity (Supplementary file 1; Table.S5 & Table 5). In two experiments, the LC₅₀ values of SpfrNPV exhibited an inverse correlation with the age of the larvae, with the highest LC₅₀ values observed for third instars. The strain, originally isolated from S. frugiperda, demonstrated high effectiveness, particularly against 2nd, 3rd and 4th instar larvae. The calculated LC₅₀ values were 2.6 × 10⁶, 3.9 × 10⁷ and 3.1 × 10⁸ POB’s/ml for 2nd, 3rd and 4th larval instar, respectively.