Novel alphabaculovirus isolated from Helicoverpa armigera (Lepidoptera: Noctuidae) in Iran: characterization and pathogenicity

Introduction

Helicoverpa armigera Hübner is one of the main constraints to the production of several economically important crops worldwide. The host range and trophic preference of the pest vary in different world regions (CABI, 2014; Riaz et al., 2021; Haile et al., 2021; Anonymous, 2025). In Iran, H. armigera is considered a key pest of tomato and it causes damage primarily to maize, soybean, pea, and cotton crops (Avand-Faghih et al., 2021). The complexity of H. armigera's life span, including reproduction, dispersal, host range, and adaptability to adverse environmental conditions, necessitates the implementation of an efficient integrated pest management (IPM) strategy to reduce crop losses (Riaz et al., 2021). This IPM strategy relies on the application of several pest control measures including cultural practices, synthetic and microbial pesticides, pytopesticides, host plant resistance and genetically modified organisms (GM crops) expressing a microbial toxin or interference RNAs complementary to key genes crucial for complementation of the pest life cycle (Riaz et al., 2021; Afrazeh & Jalali Sendi, 2025; Anonymous, 2025). However, extensive application of some of these control approaches, particularly chemical pesticides and GM crops have resulted in the emergence and prevalence of pesticide-resistant population of the pests (Ahmad et al., 2019; Gutierrez-Moreno et al., 2019; Legan et al., 2024; Posos-Parra et al., 2024a,b; Holman et al., 2025), commonly by changing their life history parameters (Liu et al., 2022; Hasnain et al., 2023; Ahmad et al., 2025; Lu et al., 2026). Alterations such as loss of functions of transporter/receptor proteins, detoxification of chemical insecticides by the host enzymatic activity, regulation of gene expression, horizontal gene transfer, and symbiont microbiome in insect gut have also been shown in pesticide-resistant populations of several insects (Riaz et al., 2021; Zheng et al., 2024; Al Naggar et al., 2025; Ahmad et al., 2025; Lu et al., 2026). Therefore, intelligent and environmentally safe pest control measures within the context of sustainable agriculture have been the subject of numerous research studies. Host-specific biocontrol agents, including bacteria, fungi, nematodes, viruses, and botanical-based insecticides, and particularly highly virulent variants/strains of insect pathogens, have been suggested as the most promising alternatives to chemical pesticides (Yang et al., 2024; Afrazeh & Jalali Sendi, 2025; Holman et al., 2025).

The insecticidal property of baculoviruses (family Baculoviridae), a group of viral pathogens of arthropods, in terms of specificity, virulence, and safety, has been widely studied since the last decades (Arrizubieta et al., 2013, 2015; Magholifard et al., 2014, 2017; Eroglu et al., 2018; Costa et al., 2019; Martinez-Castillo et al., 2022; Kenis et al., 2023; Pandi et al., 2024; Yang et al., 2024; da Silva et al., 2025; Garcia-Munguia et al., 2025). The Baculoviridae family is divided into four genera, including Alpha-, Beta-, Gamma-, and Delta-baculoviruses (Simmond et al., 2024). Baculoviruses belonging to the genus Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPVs) comprise pathogenic isolates with a restricted and narrow host range and infect only closely related insect species pests such as Anticarsia gemmetalis Hübner and Cydia pomonella Linnaeus (Clem & passarelli, 2013; Costa et al., 2019). Virions of NPVs contain morphologically single (SNPV) or multiple nucleocapsids (MNPV), and in both cases, many occluded virions (ODVs) are occluded within each occlusion body (OB). The MNPVs have been isolated only from Lepidoptera. It is suggested that the MNPV phenotype has an advantage over the SNPV due to accelerated primary infection and systemic progression (Washburn et al., 2003). Phylogenetic classification of baculoviruses has been mainly based on the sequences of their conserved genomic domains such as late expression factor 8 (lef-8), late expression 9 (lef-9) and polyhedrin (polh) genes (Jehle et al., 2006). The biocontrol properties of Baculovirus heliothis was first reported from China in 1975 (Ignoffo, 1999). Subsequently, NPVs isolated from cotton bollworm in several countries, such as Brazil (Costa et al., 2019; da Silva et al., 2025), Türkiye (Eroglu et al., 2018), Iran (Mehrvar et al., 2008a; Shahbazi et al., 2020), and the Iberian Peninsula (Figueiredo et al., 2009), and in-depth studies suggested their potential as ecologically safe pesticides for commercial production (Baillie & Bouwer, 2012; Garcia-Munguia et al., 2025). In addition, NPVs isolated from other species of Noctuidae, including Spodoptera frugiperda in Colombia (Barrera et al., 2011), the Philippines (Lavina et al., 2001), China (Lei et al., 2020), Mexico (Martinez-Castillo et al., 2022), India (Pandi et al., 2024), and from S. exigua in Mexico (Zamora-Aviles et al., 2017) have been reported. It is worth noting that nowadays genotypic variants from a significant number of naturally occurring HearNPVs have been identified, biologically purified, and some variants have been used for high-scale production of commercial biopesticides (Munoz & Caballero, 2000; Rowley et al., 2011; Barrera et al., 2011; Baillie & Bouwer, 2012; Arrizubieta et al., 2015; Costa et al., 2019; Lei et al., 2020; Shahbazi et al., 2020; da Silva et al., 2025).

Generally, native isolates or genotypic variants of NPVs are more effective at managing local insect populations than exotic isolates (Luna-Espino et al., 2018; Garcia-Banderas et al., 2020; Lei et al., 2020; Martinez-Castillo et al., 2022; Pandi et al., 2024). Indeed, the application of exotic isolates or genotypic variants of NPVs may cause adverse effects on local isolates of the viruses (Munoz et al., 1998; Munoz & Caballero, 2000) and useful insects, whereas adaptability, effectiveness, and resilience of native isolates against local pest populations have been reported (Barrera et al., 2011; Luna-Espino et al., 2018; Lei et al., 2020; Martinez-Castillo et al., 2022; Pandi et al., 2024). In fact, identifying indigenous isolates with high virulence across different ecological regions is necessary. In addition, the simultaneous presence of genotypic variants with different pathogenic properties has been reported in field and/or laboratory isolates of NPVs (Baillie & Bouwer, 2012; Shahbazi et al., 2020). Therefore, accurate characterization of genotypic variant mixture of viral isolates and biological purification are critical for host-virus interactions. Although the HearNPV isolates have been reported from several pest species feeding on tomato, cotton and tobacco in Iran, and are applied to pure and practical studies thereof (Mehrvar et al., 2008a, b; Mehrvar, 2009, 2013; Mehrvar & Saber, 2022; Kalantari et al., 2013, 2018; Allahyari et al., 2019, 2020; Magholifard et al., 2014, 2017; Gifani et al., 2015; Shazdehahmadi et al., 2016; Moshtaghi et al., 2013; Shahbazi et al., 2020; Valizadeh et al., 2020, 2024); however, their characterization at the species levels (HearMNPV/HearSNPV) has only been carried out in a few numbers of reports (Shahbazi et al., 2020; Mehrvar & Saber, 2022; Valizadeh et al., 2024). In this study, we describe the isolation of a HearNPV from a natural population of Helicoverpa armigera larvae from tomato crops in East-Azarbaijan province, Iran, which we designated H. armigera nucleopolyhedrovirus-Iran (HearNPV-IR). Microscopic properties clarified this wild-type isolate. Further molecular dissections using genomic approaches targeting highly conserved Baculovirus domains, such as the polh, lef-8, and lef-9 sequences, and phylogenetic studies placed the HearNPV-IR isolate within the HearMNPV clade. Biological studies of this isolate against the third-instar larvae of H. armigera showed that the HearMNPV-IR isolate can be introduced as promising biocontrol agent for appropriate control strategies of H. armigera in the future.

Materials and methods

Insect rearing

The H. armigera colony was established with pupae received from Tarbiat-Modares University, Tehran, Iran. It was maintained in a growth chamber at constant environmental conditions (25±1°C, 70±5% RH and a photoperiod of 16:8 h light: dark) and reared on a pinto bean-based semisynthetic diet (Naseri et al., 2009). To avoid covert infections (da Silva et al., 2025), the colony was checked by polymerase chain reaction (PCR) amplification of the viral polh gene (Shahbazi et al., 2020).

Virus isolation, purification, and propagation

Two NPV isolates were originally obtained from Helicoverpa armigera larvae from traditional tomato fields located in East-Azarbaijan province (38°05' N and 46°46' E), Iran, in 2014. Larvae were individually stored in 1.5 ml plastic micro tubes at -20 °C in the Seed and Plant Certification and Registration Research Institute, Karaj, Iran. Occlusion bodies (OBs) were extracted from cadavers, and the fresh OB stocks for each isolate were obtained by multiplying them in newly molted 5th instar larvae of H. armigera using the plug-diet method as described elsewhere (Shahbazi et al., 2020). Briefly, groups of 40 fifth-instar overnight-starved larvae were allowed to feed from a diet plug inoculated with 5×105 OB/ml. After 24 h, the larvae were transferred to individual cups containing virus-free diet and reared under standard conditions until death. Occlusion bodies from cadavers were purified as described above.

Microscopic studies

Visualization and quantification of viral OBs were carried out under 400-1000× magnification by light microscope (Micros, Austria) and a Neubauer hemocytometer. The refractive protein crystals of OBs were visible under phase contrast illumination of the light microscope. The visibility of the crystalline structures was further enhanced by staining the OBs with methylene blue. For this purpose, the appropriate volume of OBs suspension was mixed with diluted methylene blue. A volume of 10 µl of the stained OB suspension was loaded into both halves of the hemocytometer using a pipette and allowed to stand for 15 min to facilitate sedimentation of the particles onto the chamber floor, where the bright OB structures were clearly visible.

Isolation of viral DNA and preliminary detection of virus

Purified viral OBs, extracted from individual larvae, were used to extract viral genomic DNA (gDNA) using the SinaPure Viral Kit (SinaClone, Iran) according to the manufacturer's instructions. The extracted DNA was quantified using a spectrophotometer (NanoDrop ND-1000 UV/Vis, USA). Further confirmation of the virus in both isolates was performed by PCR amplification of the viral polh gene as a marker (Christian et al., 2001; Jehle et al., 2006; Shahbazi et al., 2020).

Phylogeny and Kimura-2 parameter analysis of virus isolate

Partial single and concatenated sequences of the polh, lef-8, and lef-9 genes (Jehle et al., 2006) of an isolate (hereafter HearNPV-IR) were targeted for the construction of a phylogenetic tree. Amplification of these core genes was performed by PCR using the following primer sets (Table 1): rPol-f/rPol-r (Christian et al., 2001), prL8.1/prL8.2, and prL9.1/prL9.2 (Lange et al., 2004). PCR products were purified using a gel DNA recovery kit (Vivantis, South Korea) and sequenced (Bioneer, South Korea). Multiple-sequence alignments of single sequences of the three genes and their concatenated sequences, treated as a single sequence, were performed using the MUSCLE program (Nai et al., 2017). The phylogenetic tree was constructed using the minimum-evolution (ME) method in MEGA6 (Lange et al., 2004; Jehle et al., 2006). Confidence levels for the branching points were determined using 1000 bootstrap replicates. Distance matrices between HearNPV-IR and other closely related species were determined for partial lef-8, lef-9, and polh gene sequences using the pairwise distance calculation in MEGA version 6.0, applying the Kimura-2-parameter (K-2-P) model. Homologous sequences of H. armigera GV were also included in the K-2-P analysis.

Biological activity of virus isolates against laboratory colony

Concentration-mortality response (LC₅₀) and time-mortality of HearMNPV-IR were performed by using seven different viral concentrations (3×103to 3×109 OBs/ml) in 3rd instar larvae of H. armigera. Bioassays were carried out in two separate experiments, each with three replications of 10 third-instar larvae. The 3rd instars newly molted and overnight-starved were allowed to feed from a diet plug inoculated with the virus isolate at the following concentrations (3×103, 3×104, 3×105, 3×106, 3×107, 3×108, and 3×109 OBs/ml/larvae). After 24 h and feeding the whole diet, the larvae were transferred to new individual trays containing a virus-free diet. Sterile water containing food dye, at the same concentration as used for virus-contaminated diet, was applied on diet and used as negative control. Larvae were incubated in a growth chamber under constant environmental conditions (26 ± 1 °C, 70 ± 5% RH, and a photoperiod of 16:8 h light: dark), and mortality was recorded at 10 days post-infection (dpi) in 24 h intervals. Baculovirus infection in dead larvae was diagnosed by syndrome assessment, including "wilt" and integument discoloration, and by light microscopy. The mean larval mortality at each concentration was subjected to probit analysis to determine the median lethal concentration (LC₅₀) using the POLO-PC program (Le Ora Software, 1987). Time-to-mortality results for larvae were analyzed using Kaplan-Meier estimates (Kaplan & Meier, 1958) for survival curve analysis in SPSS statistical program 26 (Anonymous, 2019).

 Results

Microscopic structure and virus detection

The refractile protein crystals of the HearNPV-IR isolate were observed under phase-contrast illumination. The polyhedral structure and irregular shape of OBs were obvious at 400× and 1000× magnification under light microscopy. The polh gene sequence derived from the amplified fragment by universal primer set (rpol-f/rpol-r) confirmed NPV infection (Fig. 1).

Phylogenetic and Kimura-2 parameter analysis of virus isolates

Partial sequences of highly conserved genes of polh, lef-8, and lef-9 among baculoviruses were used as targets for PCR to characterize lepidopteran NPVs. Amplified PCR products from both isolates were directly sequenced. Comparison of the amplified sequences from both isolates revealed 100% identity across all three genes (data not shown). Therefore, one sequence of each gene for HearNPV-IR was deposited in GenBank (NCBI) under the accession numbers OP751377 (polh), OP571375 (lef-8), and OP751376 (lef-9). Phylogenetic trees were constructed based on single and concatenated partial sequences of lef-8, lef-9 and polh genes to determine the taxonomic relationship between the newly detected HearNPV-IR isolate and closely related NPVs (Table 2). The results indicated that HearNPV-IR clustered with other HearMNPV isolates and was located close to the H. armigera MNPV Russia isolate 3154 in all phylograms (Fig. 2). To precisely clarify the relationship of Helicovrapa-derived NPVs, the K-2-P distance between the aligned polh, lef-8 and lef-9 nucleotide sequences was performed (Table 3); if the value of the nucleotide locus distance between two NPVs is less than 0.015, they are considered the same species, but a distance of more than 0.050 defines those isolates as different species. Additionally, for the K-2-P values between 0.015 and 0.05, more complementary data is needed to determine the viral species (Jehle et al., 2006; Nai et al., 2017). The distances between HearNPV-IR and other HearMNPVs were less than 0.015 for single lef-8 and lef-9 sequences, but only the polh sequence distance was equal to 0.015. The distances between HearNPV-IR and other HearMNPVs were less than 0.015, whereas the distances for all three genes between HearNPV-IR, the SNPVs group, and HearGV exceeded apparently much more than 0.050. Therefore, based on the data presented in Table 3, the HearNPV-IR isolate belongs to the HearMNPV group.

Table 1. Primers used for amplification of NPVs conserved genomic regions from Helicoverpa armigera

Biological activity of HearNPV-IR isolate against H. armigera

The infectivity of the HearNPV-IR isolate was studied on freshly molted 3rd-instar larvae of the H. armigera laboratory colony under controlled growth chamber conditions. To test the time response of H. armigera, a survival-time analysis was conducted on 3rd-instar larvae inoculated with different concentrations of OBs from HearNPV-IR. Survival began to decline at 2 dpi in the three higher OB concentrations, and 100% mortality was observed in 3rd instar larvae at 5 to 7 dpi. The median survival time (ST₅₀) was 3 and 4 dpi for larvae receiving OBs at concentrations equivalent to 3×109 and 3×108 OBS/ml, respectively. The survival curve reached ST₅₀ at approximately 144 h after inoculation for the fourth (3×106) and fifth (3×105) dilutions of polyhedral suspension. All larvae were alive at 10 dpi in the control treatments (Fig. 3).

Discussion

Naturally occurring pathogenic baculoviruses of H. armigera could provide valuable sources for manufacturing and improvement of local microbial insecticides. Elcar, a Heliothis virus-based product, was the first baculovirus-derived biocontrol agent registered in 1975, although it was not continued (Mondal et al., 2021). More recently, several indigenous variants of H. armigera NPV identified from China (HearNPV-C1, HearNPV-G4), Spain (HearNPV-Sp1), Africa (HearNPV-NNg1) and Australia (HearNPV-Aus, HearNPV-AC53) as promising candidates for the development of local biopesticides against native insect hosts (Arrizubieta et al., 2015; Williams et al., 2022; Kenis et al., 2023; Garcia-Munguia et al., 2025).

Fig. 1. Microscopic and molecular detection of unknown NPV from Helicoverpa sp. dead larvae. (A) Microscopic observation of liquefaction extracted from Helicoverpa armigera larvae at 1000× magnification. Black arrows indicate the polyhedral occlusion bodies (OBs) of the virus. (B) PCR detection of partial polyhedrin gene (400 bp) by universal primer pair. M indicates DNA ladder 1Kb (Fermentas) and Autographa californica multiple nucleopolyhedrovirus (M25054) and HearNPV-200IR_az (MN840078) are positive controls. ddH₂O represents negative control of PCR.

Table 2. Accession numbers for the lef-8, lef-9, and polh genomic sequences of several HearMNPV and HearSNPV isolates retrieved from GenBank and used for phylogenic studies

It is known that the application of exotic commercial products originated from exogenic virus variants may cause adverse effects on the biological control of local host biotypes (Munoz et al., 1998; Munoz & Caballero, 2000). However, this phenomenon is important for reducing the overall pathogenicity of virulent variants and preventing epizootic development, as shown by the case of defective variants US2C and US2E in the Spod-X commercial biopesticide against Spodoptera exigua in the USA (Munoz & Caballero, 2000). Additionally, this phenomenon enables access to NPVs for subsequent transmission cycles, a phenomenon known as biopesticide sublethal effects. In addition, native host biotypes may show varying susceptibility to viral OBs originating from different geographic niches (Figueiredo et al., 2009).

Fig. 2. Phylogenetic position of HearNPV-IR among the other closely related NPV isolates. Minimum evolution phylogram is based on partial sequences of polh (A), lef-8 (B), lef-9 (C) genes and their concatenated sequences (D). Numbers at the nodes indicate bootstrap value analyses.

 Table 3. Pairwise Kimura-2-parameter distances of the aligned partial sequences of individual polh (A), lef-8 (B), and lef-9 (C) genes of HearMNPV-IR to published sequences in

GeneBank. (A)

(B)

(C)

Therefore, isolation of local wild-type viruses, in vivo/in vitro purification of their genotypic variants, comparative pathogenicity evaluation of variants, and selection of variants with suitable insecticidal properties against indigenous pests are necessary for developing effective virus-based biopesticides (Lei et al., 2020; Pandi et al., 2024). In this study, two field isolates of NPV were obtained from a natural population of the invading pest Helicoverpa armigera in Iran using microscopic and molecular detection approaches. Visual observation of OBs revealed a typical polyhedral morphology with variable diameters, easily visible at 1000× magnification. However, detailed information on virus particles and morphology is typically needed, and future studies should use higher-resolution approaches such as transmission electron microscopy (TEM). This simplified diagnosis by light microscopy was also performed in several previous studies for visualization of virus structures in liquefactions from insect cadavers (Lavina et al., 2001; Grzywacz et al., 2005; Ferrelli et al., 2016; Nai et al., 2017; Eroglu et al., 2018; Costa et al., 2019). Simple and primitive detection of unknown NPVs with PCR utilizing specific primers based on polyhedrin (or granulin) gene sequences has been commonly used in laboratories (Jehle et al., 2006; Arrizubieta et al., 2013; Krejmer-Rabalska et al., 2019; Lei et al., 2020; Dou et al., 2024; Pandi et al., 2024; Erlandson et al., 2024). We amplified the polh gene fragments from gDNA of both isolates using commonly used polh gene primers (Jehle et al., 2006). Further sequencing of the amplicons confirmed that the isolated NPVs belong to the baculovirus family. Therefore, both isolates were named HearNPV-IR provisionally according to the host from which they were isolated. Further amplification and sequencing of the Baculovirus highly conserved genes lef-8, lef-9, and polh from both isolates confirmed complete (100%) sequence identity between the two isolates. Therefore, one isolate (isolate 1) was used for phylogenetic and biological studies. The Kimura-2-parameter (K-2-P) distance model is commonly used to estimate genetic relatedness and taxonomic placement among closely related NPVs (Nai et al., 2017; Lei et al., 2020; Erlandson et al., 2024). The K-2-P distances between the aligned polh, lef-8 and lef-9 nucleotide sequences indicated that the virus obtained from diseased larvae is a typical isolate of H. armigera multiple nucleopolyhedrovirus (HearMNPV).

Table 4. Summary of previous studies dealt with Helicoverpa armigera nucleopolyhedrovirus isolates in Iran

^aHaNPV: Helicoverpa armigera nucleopolyhedrovirus; HaMNPV: H. armigera multiple nucleopolyhedrovirus; HaSNPV: H. armigera single nucleopolyhedrovirus. ^bIRIPP: Iranian Plant Protection Research Institute; SPCRI: Seed and Plant Certification and Registration Research Institute. ^cPCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism; RAPD: Randomly amplified polymorphic DNA; ND: Not determined.

Fig. 3. Kaplan–Meier survival curve for Helicoverpa armigera 3^rd instar larvae after feeding on diet plug inoculated with different occlusion body concentrations of HearNPV-IR isolate at 10 days post infection.

Also, the ME phylogram was constructed from single and concatenated partial sequences of the three genes, and the clustering of HearMNPV-IR with other HearMNPV isolates confirmed the results of the K-2-P analysis. The Iranian HearMNPV-IR isolate was most closely related to the HearMNPV-3154 isolate from Russia. It has been documented that Helicoverpa NPVs obtained from different species and geographic regions are geographical variants of the same viral species (Jehle et al., 2006; Rowley et al., 2011; Arrizubieta et al., 2013). Although a significant number of biocontrol research activities using HearNPV have been carried out in Iran, studies are largely limited to either biologically uncharacterized endemic isolates or imported isolates from international research institutes/commercial companies (Summarized in Table 4). Indeed, the overall pathogenicity parameters of a single viral isolate in a specific host species is orchestrated by collaborative interactions of multiple genotypic variants, including virulent, defective, haplotype, and recombinant variants (Munoz & Caballero, 2000; Baillie & Bouwer, 2012; Shahbazi et al., 2020; Pandi et al., 2024; Garcia-Munguia et al., 2025) or even different virus species (Washburn et al., 2003; Beperet et al., 2021; Arrizubieta et al., 2022). Therefore, using genetically and biologically homogenous isolates and strains is strictly recommended for future studies.

The pathogenicity of HearMNPV-IR isolates 1 was tested against laboratory-reared, newly molted 3rd-instar larvae of H. armigera under growth chamber conditions. The LC₅₀ value of the isolate (4 x 10 ^5 OB/ml) was comparable to those reported in the screening for biological activity of HearNPV-01 by Eroglu et al. (2018) in Türkiye and in the biological control program of H. armigera using the HearSNPV-S1 isolate in Spain (Arrizubieta et al., 2013). However, compared with the infectivity of MNPVs reported from other countries, the HearMNPV-IR showed a higher LC₅₀ value. These include the former USSR HearMNPV-SP1 on Spanish H. armigera (Arrizubieta et al., 2022), Nicaraguan Spodoptera frugiperda MNPV (SfMNPV)-NIC on H. armigera from Honduras (Simon et al., 2004), and Colombian SfMNPV-COL (Barrera et al., 2013), Indian SfMNPV-NBAIR (Pandi et al., 2024), and Chinese SfMNPV-HUB (Lei et al., 2020) isolates on local S. fugiperda colonies. It is worth noting that we assayed the HearMNPV-IR on H. armigera 3rd instar larvae, whereas the 2nd instars were used in the above-mentioned reports, except for the SfMNPV-HUB, which was applied on the 4th instars of the host. Williams & Payne (1984) showed that although the LC₅₀ and LT₅₀ values of SNPVs and MNPVs are independent of larval weight, they are affected by larval stage (Williams & Payne, 1984; Magholifard et al., 2014). Therefore, our study on the host 3rd instars could somehow explain the high LC₅₀ value of HearMNPV-IR. Survival time and mortality response of treated larvae are critical parameters to assess the effectiveness and virulence of virus isolates. Surprisingly, the LT₅₀ value of HearMNPV-IR (72-144 h) was shorter than those of SfMNPV-NIC (131 h), SfMNPV-COL (167 h), and HearMNPV (143 h) (Simon et al., 2004; Barrera et al., 2013; Arrizubieta et al., 2022). Unlike the single nucleopolyhedrovirus envelopes that contain a single rod-shaped virus particle, multiple nucleopolyhedroviral particles are enveloped in groups that could carry different genotypic variants of a certain viral species or even different species of viruses, particularly in mixed-infected host insects (Wahburn et al., 2003; Beperet et al., 2021; Arrizubieta et al., 2022).

Also, compared to HearMNPVs, HearSNPVs display lower LC₅₀ and LT₅₀ values and higher OB production rates (Luna-Espino et al., 2018; Lei et al., 2020; Garcia-Munguia et al., 2025). Arrizubieta et al. (2022) showed significant enhancement in the pathogenicity parameters of HearMNPV in larvae of H. armigera, S. frugiperda, and Mamestra brassicae when coinfected with HearSNPV. Further quantitative PCR (qPCR) analysis revealed a small fraction of HearSNPV genome compared to that of HearMNPV (Arrizubieta et al., 2022). The presence of HearSNPV isolates circulating in tomato fields of distinct geographical regions, including the North West of Iran, where the HearMNPV-IR was isolated, has been reported in our previous study as well (Shahbazi et al., 2020). Therefore, although we did not purify the HearMNPV-IR isolate by in vitro or in vivo procedures, the presence of genotypic variants or other NPV species (e.g., HearSNPV) that confer a fast-kill phenotype (lower LT₅₀) could not be ruled out. It is well known that the biological activity of the NPV species isolated from different geographic areas differs from each other in terms of LC₅₀, LT₅₀, and/or OB production rates within the host body (Luna-Espino et al., 2018; Lei et al., 2020; Garcia-Munguia et al., 2025). Also, the virulence of a single isolate differs from that of single genotypic variants or experimental mixtures of selected variants of the same isolate (Luna-Espino et al., 2018; Lei et al., 2020; Martinez-Castillo et al., 2022; Pandi et al., 2024). The latter is well-defined for the two defective variants, US2E and US2C, in the genotypic variant composition of the SeMNPV-US2wt field isolate used for commercializing Spod-X biopesticide (Munoz & Caballero, 2000). Taking all together, further purification of HearMNPV-IR isolate is necessary to elucidate its genotypic variants composition and pathogenic parameters. In summary, the HearMNPV-IR was clearly identified as a pathogenic, fast-killing isolate, demonstrating a desirable lethal concentration and rapid kill rate against the 3rd instars of the pest. Therefore, it offers valuable opportunities for future detailed studies and for its use as an active bioagent in developing virus-based biopesticides for successful biological control of H. armigera in the Middle East, particularly in Iran. Although the sequences of the Baculovirus conserved genes polh, lef-8, and lef-9 showed no detectable differences between the two HearMNPV isolates (1 and 2) in this study, future investigations using whole-genome sequencing and comprehensive comparisons of their biological activities are strongly recommended.

Author's Contributions

Raheleh Shahbazi: Investigation, Data analysis, Writing original draft. Masoud Naderpour: Conceptualization, Supervision, Methodology, Formal analysis, writing original draft, Reviewing and editing manuscript, Project management and funding acquisition. Reza Talaei-Hassanloui: Methodology, Formal analysis, Reviewing manuscript.

Author's Information

Funding

This work was supported financially by the Vice-Presidency for Science and Technology (VPST, Grant number 11/59717), SPCRI and AREEO (Grant number 14-08-08-9451).

Data Availability Statement

All data supporting the findings of this study are available within the paper. The specimens examined in this study are deposited in the first author's collection at the Seed and Plant Certification and Registration Research Institute, Karaj, Iran and are available by the curator upon request.

Acknowledgments

We thank Dr. Sorena Sattari, former Vice-Presidency for Science and Technology; Dr. Mohammad Hassan Asareh, former General Director of Seed and Plant Certification and Registration Research Institute (SPCRI); and Dr. Eskandar Zand, former General Director of Agricultural Research, Education and Extension Organization (AREEO), Iran, for providing the financial and infrastructure supports.

Ethics Approval and Consent to Participate

Insects were used in this study. All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by the author.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Generative AI statement

The authors declare that no Generative AI tools were used in the writing, analysis, or preparation of this manuscript.