Genomic characterization of a baculovirus isolate, Malacosoma neustria nucleopolyhedrovirus Eriogaster neogena isolate (ManeNPV-Er) from Mongolia

Abstract

Background

Baculoviruses are highly diverse insect-specific DNA viruses that have coevolved with their lepidopteran hosts, often exhibiting high host specificity and geographic population structures. To investigate the genomic basis of host - associated divergence, we sequenced and analyzed the complete genome of a baculovirus isolated from symptomatic Eriogaster neogena larvae collected in Mongolia.

Methods

The complete genome of this isolate, designated Malacosoma neustria nucleopolyhedrovirus Eriogaster neogena isolate (ManeNPV-Er), was sequenced and comparatively analyzed. Phylogenetic and pairwise K2P distance analyses based on lef8/lef9/polh were performed to evaluate its taxonomic relationship with other baculoviruses.

Results

Comparative genomic analysis revealed an overall nucleotide identity of 86.9% with Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2), which was isolated from a different host species (Malacosoma neustria) in Turkey. Despite their close taxonomic relationship, this low identity indicates substantial genomic divergence. Phylogenetic and pairwise K2P distance analyses based on lef8/lef9/polh confirmed their conspecific status. However, the corrected K2P distance suggests incipient divergence, which may require additional genomic and biological evidence to confirm. The significant genetic differentiation of ManeNPV-Er is potentially driven by the combined effects of adaptation to a distinct host and geographic isolation in a different region. Such divergence patterns are consistent with the parallel diversification commonly observed between baculoviruses and their lepidopteran hosts.

Conclusions

Collectively, our findings expand the current understanding of baculovirus genomic diversity and provide new insights into how the interplay of host shift and geographic separation influences viral speciation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-026-03225-y.

Introduction

The family Baculoviridae comprises large, double-stranded DNA viruses that exclusively infect insects from the orders Lepidoptera, Hymenoptera, and Diptera. Owing to their high host specificity and safety for non-target organisms, baculoviruses have long been recognized as environmentally friendly agents for biological pest control [1]. It is characterized by a circular genome ranging from 80 to 180 kbp in size [2], enclosed within rod shaped nucleocapsids, measuring 230–385 nm in length and 40–60 nm in diameter [3]. As an ancient viral lineage, baculoviruses are believed to have co-evolved with their insect hosts [4, 5]. While some baculovirus isolates display strict host specificity, others exhibit a broader host spectrum. Among Alphabaculoviruses, both specialist and broad-spectrum strains have been described, highlighting the evolutionary and ecological significance of host range diversity. The Baculoviridae family is classified into four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus and Deltabaculovirus. Furthermore, members of Alphabaculovirus are subdivided into two groups, designated Group I and Group II, a classification primarily based on the phylogeny of the envelope fusion protein involved in budded virions for cellular entry, GP64 in Group I and F protein in Group II [6].

Within the family Lasiocampidae (Lepidoptera), several genera contain defoliating species that hold both ecological and economic importance. Among these, Malacosoma neustria and Eriogaster neogena are widespread polyphagous defoliators, with distributions spanning Western Europe and Eastern Asia [7]. Eriogaster neogena is a characteristic species of desert-steppe ecosystems. Its larvae have been reported to develop on plants of the genera Caragana (Zolotuhin, 1995) and Astragalus (Gorbunov, 2011). The larvae of these species are notable for their gregarious behavior, construct silk tents on host plant branches [8]. This shelter-building habit, which is absent in certain Malacosoma species, often renders conventional insecticide sprays ineffective against the protected larval colonies.The Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2) was previously isolated from M. neustria larvae reported from in Turkey [9]. In the present study, we describe a closely related baculovirus isolated from E. neogena larvae collected in Mongolia. Although both isolates may fall within the same species according to current ICTV criteria, host-associated and geographic genetic variation are biologically significant and may indicate early stages of adaptive divergence. Given their distinct geographic distributions and differences in pest status host ranges and ecological niches, such ecological factors likely influence virus-host contact rates and impose distinct selection pressures, potentially driving genomic differentiation among baculovirus isolates. Therefore, whole-genome comparison between the Mongolian isolate (ManeNPV-Er) and the Turkish isolate (ManeNPV-T2) offers a valuable opportunity to elucidate how geographic and host factors shape baculovirus genome architecture and pathogenic characteristics.

Baculoviruses represent one of the most diverse groups of large double-stranded DNA viruses infecting arthropods, particularly Lepidoptera. Their coevolution with specific hosts and adaptation to distinct ecological niches have generated remarkable genomic plasticity and host specificity. Previous studies have demonstrated that closely related baculoviruses can exhibit parallel diversification patterns that mirror those of their hosts, implying that host-driven selection and geographic separation are important forces shaping baculovirus evolution [10]. Understanding such patterns of divergence is essential not only for clarifying taxonomic boundaries within the Baculoviridae family but also for improving the efficacy and safety of baculoviruses as biological control agents in integrated pest management programs.

To investigate these host-associated genomic differences, we sequenced and analyzed the complete genome of a baculovirus isolated from symptomatic E. neogena larvae in Mongolia. Host identification was confirmed through morphological characteristics and cytochrome c oxidase subunit I (COI) barcoding. The resulting isolate, designated ManeNPV-Er, possesses a genome of 129,114 bp. Comparative genomic analysis revealed an overall nucleotide identity of 86.9% with Malacosoma neustria nucleopolyhedrovirus isolate ManeNPV-T2, indicating substantial genomic divergence between the two isolates despite their close taxonomic relationship. Notably, we identified 6 open reading frames (ORFs) unique to ManeNPV-Er that have no detectable homologs in ManeNPV-T2, suggesting that host species differences and/or geographic isolation have contributed to genotypic divergence with potential implications for host range and virulence. Such divergence patterns are consistent with the co-diversification observed between baculoviruses and their lepidopteran hosts, supporting the notion that host adaptation plays a key role in shaping baculovirus evolutionary trajectories.

Collectively, these results provide new genomic insights into baculovirus diversity infecting hosts, and they emphasize the importance of combining morphological, molecular, and genomic data when assessing baculovirus taxonomy, evolution, and application as biological control agents.

Materials and methods

Baculovirus isolation and host identification

ManeNPV-Er and its host larvae were collected in 2025 at Buutsagaan soum, Bayanhongor province, Mongolia (coordinates:46°10’06.9"N, 98°43’00.6"E, altitude 1588 m). The collection included both asymptomatic individuals and larvae exhibiting characteristic symptoms of baculovirus infection.

Genomic DNA was extracted from insect tissue using the MightyPrep Reagent for DNA (TAKARA). The COI barcode region was amplified with universal COI primers [11]. While the viral lef8, lef9, and polh genes were amplified following the previously published protocols [12]. The amplification was performed according to the manufacturer’s instructions using TransStart^® FastPfu DNA Polymerase (TransGen Biotech). The PCR conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. The amplified products were subjected to Sanger sequencing, and the resulting sequences were compared and analyzed against DNA barcode sequences available in BOLD systems [13]. Information on both primer sequences is provided in Table S1.

Electron microscopy examination

Occlusion bodies (OBs) were obtained from the cadavers of infected larvae. The purification procdure involved sequential steps of maceration, homogenization, filtration, centrifugation, and final purification via sucrose density gradient ultracentrifugation [14].

Purified OBs were examined for detailed morphological characteristics using a scanning electron microscope (SEM; Hitachi, Japan) operated at an accelerating voltage of 5 kV. The dimensions of the ManeNPV-Er OBs were measured with precision using data extracted from 50 complete SEM image samples.

Viral DNA isolation, sequencing, and assembly

The purified OBs were resuspended in an alkaline solution (100 mM Na₂CO₃, pH 10.8) and incubated at 37 °C for 30 min. The pH was then adjusted to 8.0 using 1 M hydrochloric acid. Ribonuclease A (RNase A) and Proteinase K were added, followed by incubation at 37 °C for 2 h and subsequently at 65 °C for an additional 2 h. Viral DNA was extracted using a phenol/chloroform/isoamyl alcohol (25:24:1) mixture and precipitated with ethanol. DNA concentration was measured using a spectrophotometer, and purity was assessed based on the 260/280 absorbance ratio.

Whole-genome sequencing of ManeNPV-Er was performed using the DNBSEQ platform. After quality filtering, the average read length was 150 bp, and the sequencing depth reached 9,557×. The Q20 and Q30 values were 97.54% and 92.28%, respectively. High-quality paired-end reads were assembled de novo into contigs using Unicycler (v0.5.0) [15].

Genome sequence analysis

Open reading frames (ORFs) encoding proteins of at least 50 amino acids were predicted using FGENESV [16] and the NCBI ORF Finder. For overlapping ORFs shorter than 75 base pairs, only the larger ORF was annotated unless the smaller one corresponded to a conserved baculovirus homolog [17]. This approach is based on the assumption that expression of the smaller ORF is improbable. ORFs located within homologous repeat (hr) regions were excluded from annotation due to their relative instability. The identified ORFs were annotated according to homology by the Protein-Protein BLAST algorithm.

Gene parity plots were constructed to compare ORF organization between ManeNPV-Er and selected baculoviruses [18].

Genomic homology between ManeNPV-Er and other baculoviruses was assessed using EMBOSS Stretcher [19]. hrs were identified using the tandem repeats finder [20], and EMBOSS palindrome [21] was utilized to search for palindrome sequences.

To identify early and late promoter motifs, a 180-nucleotide region upstream of each initiation codon was analyzed. The early promoter component was recognized by the presence of a TATA-box motif with a CAKT transcription start site located 25–35 nucleotides downstream [22, 23]; additionally, TATA-like elements (TAATWAA) were also detected upstream of LEF genes [24]. The late promoter element was defined as DTAAG, typically found around 60 nucleotides upstream of the ORF initiation codon [25].

Phylogeny and K2P analysis

Protein sequences encoded by the 38 core genes were obtained from 108 baculovirus genomes, including ManeNPV-Er (Table S2), and aligned using MAFFT with the auto strategy and standard alignment mode [26]. The alignments were performed in OrthoFinder (v2.5.5) with default settings [27]. Following alignment, the sequences were concatenated while maintaining the same gene order. The optimal partitioning scheme for maximum likelihood (ML) analysis was identified using RAxML-NG (v1.2.0) [28]. PartitionFinder2 was further used to determine the best ML partitioning scheme according to the Bayesian information criterion (BIC) and a greedy search algorithm with linked branch lengths [29]. The ML phylogenetic tree was then reconstructed in IQ-TREE using the Ultrafast bootstrap approach with 5000 replicates.

Kimura 2-parameter (K2P) pairwise distances [30] were computed separately from the aligned nucleotide sequences of lef-8, lef-9, and polh fragments, as well as from the 38 core gene fragments (Table S2), using the pairwise distance function in PHYLIP (v3.6). Substitution rates across sites were assumed to be uniform, and alignment gaps were treated as missing data to ensure a thorough and accurate assessment of sequence variation.

Host-virus coevolution analysis

We analyzed the phylogenetic relationships between baculoviruses and their hosts to visualize cross-species transmission and the relative occurrence of virus-host coevolution. A tanglegram was constructed by comparing the phylogenetic trees of the baculoviruses and their respective hosts. Cophylogenetic relationships among 104 baculoviruses and 67 lepidopteran hosts were assessed using the ParaFit (v0.4.2) [31] and PACo (v5.8) [32] algorithms. Host and virus phylogenies were reconstructed based on COI sequences using the ML method, and patristic distance matrices were generated with cophenetic.phylo. Host parasite associations were represented as a binary incidence matrix.

The ParaFit global test and link-level permutation test were employed to evaluate overall and pairwise cophylogenetic congruence. In parallel, PACo analyses were conducted to calculate Procrustes residuals for each host-virus association, providing further insight into link-specific coevolutionary patterns.

Data summary

The complete genome sequence of Malacosoma neustria nucleopolyhedrovirus Eriogaster neogena isolate (ManeNPV-Er) has been deposited in GenBank under accession number PX586830.

Result

Field collection and host identification

Baculoviruses and their host larvae were collected in 2025 from Buutsagaan soum, Bayanhongor province, Mongolia. The larvae were observed feeding on the narrow, spiny leaves of Caragana microphylla Lam (Fig. 1A). Morphological characteristics and mitochondrial COI gene sequencing (BIN: BOLD: AAP8259) confirmed the larvae as E. neogena. Deceased larvae exhibited typical signs of baculovirus infection, including body melanization, liquefaction, and an inverted posture hanging from branches (Fig. 1B), which is consistent with known Alphabaculovirus pathology.

Fig. 1.

Morphology of the baculovirus Malacosoma neustria nucleopolyhedrovirus Eriogaster neogena isolate (ManeNPV-Er). (A) Healthy E. neogena larva in its natural habitat. (B) A baculovirus infected E. neogena larva showing typical signs of disease, including melanization and liquefaction. (C) Scanning electron micrograph showing irregularly polyhedral occlusion bodies of the purified ManeNPV-Er

SEM revealed that the ManeNPV-Er OBs were irregularly shaped polyhedral and ranged in size from 1.02 to 2.19 μm (Fig. 1C). Measurement of 50 individual OBs yielded a mean diameter of 1.67 ± 0.26 μm, which falls within the size range previously reported for baculoviruses [3]. This result is consistent with established findings and further supports the classification of ManeNPV-Er within the genus Alphabaculovirus.

The observed size variation among the ManeNPV-Er OBs may be attributed to the virus was obtained directly from field-collected hosts, indicating that the isolate has not undergone laboratory purification to establish a genetically homogeneous viral population.

Species identification and genetic divergence of ManeNPV isolates

Species demarcation for baculoviruses relies on a combination of criteria, including host range and specificity, restriction endonuclease digestion profiles, and sequence identity among conserved genes [33, 34]. Following the approach of Jehle [12], species identification was conducted based on the concatenated nucleotide sequences of three highly conserved genes: lef-8, lef-9, and polh. K2P distances were calculated (Table 1). After concatenation, the K2P distance between the Mongolian isolate ManeNPV-T2 (GenBank accession number KY968317.1) was 0.00926, which falls below the 0.015 species demarcation threshold, indicating that both isolates belong to the same species with ManeNPV-T2. When analyzed individually, the K2P distances for lef-8, lef-9, and polh were 0.0125, 0.00403, and 0.13469, respectively. Despite the overall conspecific status, this heterogeneity, particularly in the polh gene, highlights underlying genetic variation. In accordance with these findings, the Mongolian isolate was designated as Malacosoma neustria nucleopolyhedrovirus Eriogaster neogena isolate (ManeNPV-Er).

Table 1.

Pairwise Kimura two-parameter (K2P) distances among ManeNPV-Er and related Alphabaculovirus isolates

	ManeNPV-Er	UrprNPV	CrpeNPV	HearNPV	LdMNPV	ManeNPV-T2
ManeNPV-Er
UrprNPV	0.31129
CrpeNPV	0.25668	0.29988
HearNPV	0.26625	0.30573	0.25324
LdMNPV	0.30692	0.32749	0.29159	0.30516
ManeNPV-T2	0.00926	0.31129	0.2573	0.26563	0.3088

Genome assembly and general features

Next-generation sequencing of the ManeNPV-Er isolate was performed using the DNB-seq platform, generating its complete genome sequence. The circular genome was assembled de novo, yielding a single contig representing the full viral genome. Using the ManeNPV-T2 genome as a reference, the ManeNPV-Er genome was annotated and analyzed.

The assembled ManeNPV-Er genome is 129,114 bp in length, with an overall GCs content of 38.22%, which is lower than the average GCs content reported for Group I (44.9%) and Group II (41.6%) Alphabaculoviruses [35], but comparable to that of ManeNPV-T2 (38.2%) [9]. A total of 124 open reading frames (ORFs) were annotated (Fig. 2), each with a minimum length of 50 amino acids, collectively accounting for 85.55% of the genome. Following standard convention, the polh gene was designated as ORF1, with its methionine initiation codon assigned as position zero in the physical genome map of ManeNPV-Er.

Fig. 2.

Schematic representation of the ManeNPV-Er genome. Open reading frames (ORFs) are annotated and color coded based on gene function and evolutionary conservation: red, baculovirus core genes; five shades from green to gray, ORFs with descending levels of sequence homology to ManeNPV-T2; black, repeat regions

Notably, ManeNPV-Er lacks the core gene p6.9, which is typically conserved across most baculovirus genomes. Sequence analysis also showed that the baculovirus p6.9 gene is absent in Clanis bilineata nucleopolyhedrovirus (NC_008293), Spodoptera litura granulovirus (NC_009503), Spilosoma obliqua nucleopolyhedrovirus (KY550224.1), and Trichoplusia ni granulovirus (KU752557.1). P6.9 is a small, arginine/serine/threonine-rich DNA binding protein involved in DNA condensation and nucleocapsid assembly [36]. In addition, three unique ORFs (Er_7, Er_80, Er_99) were identified (Fig. 2), suggesting possible acquisition of novel genes during host adaptation or regional diversification.

Phylogenetic analysis

The K2P distance calculated from the lef-8, lef-9, and polh genes was 0.00926, which is below the established interspecific threshold for baculoviruses (0.015–0.050). In contrast, the K2P distance derived from the 38 core genes was 0.0509, falling within the adjusted interspecific range (0.021–0.072) [37]. These results indicate that although ManeNPV-Er and ManeNPV-T2 are closely related, genetic divergence has occurred between them, likely driven by differences in geographic distribution and host specificity.

To investigate the evolutionary relationships between ManeNPV-Er and other baculoviruses, a phylogenetic tree was constructed based on the amino acid sequences of 38 highly conserved core genes from 108 baculovirus genomes (Table S2). The analysis placed ManeNPV-Er within Group II Alphabaculovirus, consistent with the presence of the F protein and the absence of the gp64 (Fig. 3). ManeNPV-Er is most closely related to ManeNPV-T2, exhibiting 86.9% nucleotide identity, in agreement with the K2P distance analysis.

Fig. 3.

Phylogenetic relationships of ManeNPV-Er within the genus Alphabaculoviruses. An unrooted phylogenetic tree was created from the concatenated 38 core genes found in 108 sequenced baculovirus genomes. The position of ManeNPV-Er is marked in red. Bootstrap support values obtained from 1,000 ultrafast bootstrap replicates are indicated at the nodes. Major clusters representing to the four baculovirus genera the (Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus) have been simplified for clarity

Comparative genomics and structural variation

Whole genome comparison between ManeNPV-T2 and ManeNPV-Er isolates revealed substantial genotypic variation, with a total of 4,884 single nucleotide polymorphisms (SNPs) identified across their genomes. Using ManeNPV-T2 as the reference, 16 ORFs exhibited no SNPs, indicating high conservation. Among these completely conserved genes, six encode hypothetical proteins, while the remaining ten participate in critical viral life processes. Notably, this conserved core includes key components required for oral infectivity and structural integrity, such as several per os infectivity factors (pif-4 [38], pif-9 [39], and pif-7 [40]), a spheroidin-like protein precursor [41] involved in midgut binding, and ac109 [42], which is essential for BV production and nucleocapsid envelopment. Furthermore, genes regulatory to viral replication and transcription, such as the ie-0 [43], the orf133-like protein [44] required for embedding virions into polyhedra, alkaline exonuclease involved in genetic recombination [45], and nicotinamide riboside kinase-1 [46] involved in NAD⁺ biosynthesis were perfectly preserved. The remaining 105 ORFs contained a mixture of synonymous and missense mutations (Table S3). Despite these variations, the majority of ORFs shared > 90% amino acid identity between the two isolates (Fig. 2). The highly conserved genes identified may play important roles in cross host infectivity and essential viral functions.

Structural variation analysis revealed that ManeNPV-Er is 1,088 bp shorter than ManeNPV-T2. Using ManeNPV-T2 as the reference, ManeNPV-Er contains a 2,952 bp insertion (positions 75,666 − 78,619), annotated as a ribonucleotide reductase small subunit homolog, and a repeat expansion (positions 73,502 − 74,967) primarily within hr regions. Gene parity plot analysis was performed to compare gene order between the ManeNPV-Er and to assess genome collinearity and evolutionary patterns (Fig. 4). Overall, the genomes of ManeNPV-T2 and ManeNPV-Er displayed high collinearity, although three small inversions were observed, reflecting localized genomic rearrangements, indicating largely conserved gene organization.

Fig. 4.

Comparative ORF similarity and genome collinearity between ManeNPV-T2 and ManeNPV-Er. The ORF content and order of the ManeNPV-Er genome (y-axis) was compared with that of Culex nigripalpus NPV (CuniNPV, Deltabaculovirus), Neodiprion sertifer NPV (NeseNPV, Gammabaculovirus), Adoxophyes orana GV (AdorGV, Betabaculovirus), Autographa californica NPV (AcMNPV, group I Alphabaculovirus), Lymantria dispar MNPV (LdMNPV, group II Alphabaculovirus) and Cryptophlebia peltastica NPV (CrpeNPV), Urbanus proteus NPV (Urpr NPV) and Malacosoma neustria NPV (ManeNPV-T2) (x-axes). Each point in the plot represented the relative position of open reading frames in the ManeNPV-Er genome with those from other baculoviruses

BLASTX comparisons revealed distinct gene content variations between the two isolates. ManeNPV-Er lacks ten genes present in ManeNPV-T2, including eight hypothetical proteins (ManeNPV_00004, _00006, _00011, _00038, _00063, _00065, _00078, _00086, _00106, and _00131), core gene p6.9 and ac52. Deletion of ac52 has been shown to disrupt normal nucleocapsid envelopment and polyhedron formation in infected nuclei [47]. Conversely, six genes were identified as isolate-specific to ManeNPV-Er relative to ManeNPV-T2. Among these, three represent the completely novel, newly annotated hypothetical proteins mentioned earlier (Er_7, Er_80, and Er_99), while two encode ribonucleotide reductase small subunit homologs (Er_82 and Er_83), which catalyze the reduction of ribonucleotides to deoxyribonucleotides and thereby ensure an adequate dNTP supply during viral DNA synthesis [48]. The remaining unique gene, Er_97, encodes a BRO family protein that contains a conserved DNA-binding domain and has been implicated in viral genome replication and regulation. In baculoviruses, BRO proteins are frequently located in genomic regions prone to rearrangement [49, 50]. Apart from these differences, the overall ORF composition of the two genomes is highly conserved.

The 180 bp upstream regions of the three unique ORFs were analyzed to identify putative regulatory motifs. In Er_80, a late promoter sequence was detected. In Er_7, both early promoter motifs and late promoter elements were present. Er_99 lacked recognizable canonical promoter motifs. These findings suggest differential regulation of unique ORFs, which may impact viral gene expression and host interactions.

Homologous repeat regions

Most baculovirus hrs contain repeated AT-rich sequences [51]. hrs are characterized by tandem repeats that include an imperfect palindromic core and are distributed at various locations throughout the genome. These regions have been proposed or demonstrated to function as origins of DNA replication and as enhancers of early baculovirus promoters.

In ManeNPV-Er, nine hr regions were identified (Fig. 5), each composed of ~ 90 bp repeat units with an imperfect 12 bp palindrome near the center. Each hr contained 1–3 repeat units. For example, hr1, located between ac146 and ac145, contained 2 repeat units; hr2, between me53 and Er_21, contained 2 repeat units; hr3, between Er_29 and p10, contained 1 repeat unit; hr4, between dbp and rcm, contained 3 repeat units; hr5, between Er_80 and Er_81, contained 2 repeat units; hr6, between Er_99 and nrk1, contained 1 repeat unit; hr7, between pif3 and pp1, contained 3 repeat units; hr8, between Er_117 and Er_118, contained 2 repeat units; and hr9, between pif2 and pkip-1, contained 1 repeat unit (Fig. 5).

Fig. 5.

Homologous repeat (hr) regions in the ManeNPV-Er genome. Sequence alignment of ManeNPV-Er hrs repeat units. The imperfect palindrome sequence was represented with Group II Alphabaculoviruses in the red box

Coevolution of viruses and hosts

To examine the evolutionary relationships between baculoviruses and their lepidopteran hosts, we performed cophylogenetic analyses involving 104 viruses and 67 hosts, with CuniNPV - Culex nigripalpus as an outgroup (Fig. 6). The ParaFit global test showed no significant overall congruence between virus and host phylogenies (p = 0.902), suggesting that strict codivergence is not predominant across the entire dataset. The PACo analysis revealed a mean link-level residual of 2.285, while the specific association between ManeNPV-Er and E. neogena showed a residual of 1.370. This result indicates a relatively stronger phylogenetic fit for this particular virus-host association compared with the average pattern observed across the dataset. However, given the non-significant global ParaFit result, this pattern should be interpreted as descriptive variation in association strength rather than evidence of codivergence.

Fig. 6.

Cophylogenetic analysis of baculoviruses and their lepidopteran hosts. The tanglegram depicts phylogenies for 104 viruses (left) and 67 hosts (right). Global congruence was not significant (ParaFit, p = 0.902). PACo analysis revealed a mean residual of 2.285, yet the link between ManeNPV-Er and E. neogena (red) had a lower residual (1.370)

Discussion

In this study, we characterized an Alphabaculovirus, ManeNPV-Er, isolated from E. neogena larvae collected in Mongolia. Comprehensive phylogenetic analyses revealed that ManeNPV-Er firmly within Group II Alphabaculovirus, represents a variant of ManeNPV-T2, with notable genetic divergence and host specificity. These findings expand the known diversity of Malacosoma infecting baculoviruses and provide new insights into their evolutionary trajectories and host virus co-adaptation.

Species identification of baculoviruses has traditionally relied on the comparison of conserved gene sequences, particularly the lef8/lef9/polh genes, which together provide a robust framework for species delimitation. The concatenated K2P distance of 0.00926 between ManeNPV-Er and ManeNPV-T2, indicating that both isolates belong to the same species [12]. This conspecific assignment is firmly supported by the concatenated K2P matrix, which buffers against single-gene evolutionary anomalies; indeed, the relatively low whole-genome identity (86.9%) and the elevated individual polh K2P distance (0.13469) collectively reflect intense localized selection pressures and independent lineage-specific divergence rather than distinct species identities. However, the corrected K2P distance (0.0509) calculated from 38 core genes supports the notion that ManeNPV-Er may have undergone genetic differentiation, possibly driven by geographical isolation and host specific adaptation [37].

The ManeNPV-Er genome exhibits a high degree of overall collinearity with ManeNPV-T2, reflecting evolutionary stability of the Alphabaculovirus genome organization. Nonetheless, 4,884 SNP and a 1,088 bp size reduction were identified, together with local inversions and repeat expansions in hr regions. Such variation may influence replication efficiency and transcriptional regulation [52].

Functionally, the lack of a canonical p6.9 could theoretically hinder tight viral DNA packaging, potentially leading to reduced nucleocapsid stability, altered BV titers, or variations in OB morphology [36]. However, previous knockout studies in other baculoviruses have shown that while p6.9 mutants exhibit defects in viral replication, they often retain partial assembly capabilities, suggesting that the functional loss of this gene is not universally lethal [53]. The absence of p6.9 is rare among baculoviruses, though previously reported in several other species, suggesting functional redundancy or compensatory mechanisms mediated by alternative basic proteins. In these specific lineages, the phenotypic consequences of p6.9 omission appear to be offset by alternative basic proteins or functional compensation through adjacent arginine/serine/threonine-rich ORFs that mimic the protamine-like characteristics of P6.9 [54]. This loss highlights an unexpected degree of genomic plasticity among Alphabaculoviruses and underscores how compensatory mechanisms or functional redundancy can sustain core replication fitness during host-driven regional diversification. Furthermore, the deletion of ac52, a gene implicated in normal nucleocapsid envelopment, may contribute to subtle structural or replicative differences between ManeNPV-Er and ManeNPV-T2. Such gene loss highlights the ongoing genomic plasticity within Alphabaculoviruses and may underlie host adaptation or ecological specialization.

Despite the close phylogenetic affinity between ManeNPV-Er and ManeNPV-T2, the pronounced differences in the geographical range and feeding preferences of their hosts indicate long-term host separation. Such ecological divergence may have reduced opportunities for cross-infection and contributed to independent viral diversification over time. Although the global cophylogenetic analyses did not support significant overall host-virus congruence, the observed association pattern is consistent with the possibility that host-related ecological factors influence baculovirus diversification. Similar host-associated evolutionary patterns have been reported in other lepidopteran-baculovirus systems [6, 55], although the relative contributions of host adaptation, ecological isolation, and stochastic evolutionary processes remain difficult to disentangle.

Conclusions

This study enhances our understanding of the genetic and evolutionary diversity of ManeNPV. ManeNPV-Er exemplifies how geographic variation, gene gain/loss events, and host–virus coevolution collectively drive diversification within the Baculoviridae. Comparative genomic and phylogenetic analyses revealed both conserved core gene architecture and isolate-specific variations, reflecting adaptive responses to distinct ecological contexts and host associations.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

lef8

Late expression factor 8

lef9

Late expression factor 9

polh

Polyhedrin

COI

Cytochrome C oxidase

BOLD

Barcode of life database

NCBI

National center for biotechnology information

Author contributions

QQ and HZ conceptualization and supervision. QQ and HL contributed to funding acquisition and project coordination. WJ and UG data curation and formal analysis. WJ, JZ and QM Software and validation. UG, HL, BM, HW, and EB provided resources. WJ visualization. UG and WJ writing the original draft, review, and editing. All authors have read and approved the final version of the manuscript.

Funding

This research was funded by National Key Research and Development Program of China (Grant number 2022YFD1400700), National Natural Science Foundation of China (Grant number 32261143728).

Data availability

GenBank accession number: PX586830.

Declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare no competing interests.