A new nucleopolyhedrovirus isolated from a laboratory population of Cydia pomonella exhibits excellent biological activity

A new nucleopolyhedrovirus isolated from a laboratory population of Cydia pomonella exhibits excellent biological activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A new nucleopolyhedrovirus isolated from a laboratory population of Cydia pomonella exhibits excellent biological activity Yuxi Liu, Huanjuan Zhao, Bokun Wang, Yu Fei, Bing Bai, Ping Gao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9410995/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While naturally occurring insect-pathogenic viruses are often highly pathogenic and utilized as biocontrol agents, the potential of laboratory-maintained viruses remains underexplored. We report a novel nucleopolyhedrovirus, CypoNPV, exhibiting excellent biologically activity against its host, Cydia pomonella . CypoNPV is genetically similar to Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which also infects C. pomonella . Reducing viral copy numbers with formaldehyde did not enhance host population development. However, proliferated CypoNPV maintained high pathogenicity against newly hatched C. pomonella larvae. Field trials confirmed its excellent efficacy, comparable to the chemical insecticide lambda-cyhalothrin. Furthermore, CypoNPV displayed robust thermal and UV resistance; virulence was unaffected by 40°C treatment. Exposure to 60°C or UV (267 µW/cm²) for 1–4 hours decreased larval mortality to 30.00%-76.67% and 10.00%-43.33%, respectively. These findings enhance our understanding of baculovirus persistence in insects and their coevolution, thereby establishing a new avenue for the discovery and application of pathogenic viruses for pest biological control. Biological sciences/Microbiology Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Virus-host interactions represent an evolutionary arms race, where reciprocal selection pressures for pathogenicity and resistance dictate ecological outcomes, ranging from mutualism to lethality 1 . Insect-pathogenic viruses are ubiquitous in natural insect populations, with baculoviruses being the most extensively studied due to their high host specificity, potent pathogenicity, and long-standing significance in biological control 2 . This association has been recognized for over a century: polyhedrosis in silkworm was documented in the mid-19th century, and the silkworm-baculovirus system subsequently became a well-established model for insect-virus interactions 3 , 4 . In orchards, Cydia pomonella granulovirus (CpGV) serves as a benchmark, identified from naturally infected codling moth larvae and developed into highly effective products for pest management 5 – 7 . Similarly, Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) was widely deployed against the velvetbean caterpillar in Brazilian soybeans, regarded as one of the most successful viral bioinsecticides developed 8 . Commercial applications of heliothine baculoviruses further underscore the practical value of insect viruses, Helicoverpa zea SNPV formed the basis of Elcar, and Helicoverpa armigera SNPV/HearNPV became a key commercial insecticide for bollworm control in China 9 , 10 . Their utility extends beyond annual crops, with Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) developed as Gypchek for operational management in forest ecosystems 11 , 12 . More recently, Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) has been applied to management, with registered products like Cartugen and Fawligen, and ongoing research supporting its biological activity and practical potential 13 , 14 . These cases collectively demonstrate the proven biological activity and, in several instances, the clear practical and commercial value of insect pathogenic viruses. Viruses detected in laboratory colonies, rather than as overt disease outbreaks, often exist as covert, persistent, low-level, or asymptomatic infections 15 . These infections can remain undetected for extended periods, becoming apparent only under specific physiological or environmental conditions 16 . Furthermore, viral infections do not negate the biological relevance of laboratory populations. On the contrary, baculovirus covert infections are prevalent in laboratory insect populations, and can be maintained through various transmission routes 15 . The hidden persistence of these viruses suggest that indoor insect colonies may represent an overlooked source of candidate viruses for future biological control. This potential is not merely theoretical; a covert infection of Phthorimaea operculella granulovirus in a laboratory colony demonstrated stable persistence and influenced host population dynamics 17 . Latent SpltNPV was also identified in apparently healthy laboratory Spodoptera litura population and could be reactivated following heterologous virus challenge 18 . Similarly, a novel alphabaculovirus isolated from a laboratory-reared Cryptophlebia peltastica population exhibited virulence against its native host and was proposed as a potential biopesticide; and a subsequent study confirmed it high infectivity to C. pomonella 19 , 20 . Even in the case of C. pomonella , a granulovirus from a British Columbia laboratory colony was found to be identical or nearly identical to orchard isolates 21 . These findings imply that long-term laboratory insect colonies may serve as an underexplored reservoir for candidate insect viruses. While pathogenicity is a prerequisite, it alone does not suffice to confirm the applied value of these viruses 22 . Solar ultraviolet (UV) radiation rapidly degrades the efficacy of baculovirus on exposed surfaces, and temperature exerts a significant influence on viral activity, production, and persistence 23 . Therefore, a thorough evaluation of thermal stability, in conjunction with UV tolerance, is essential for assessing the realistic field potential of a laboratory-derived isolate 23 – 25 . Nevertheless, it remains largely unclear whether viruses persisting in laboratory colonies can be isolated as biologically active agents and whether they retain sufficient pathogenicity and environmental stability for practical application. This study presents the first report in insects of a novel nucleopolyhedrovirus (CypoNPV) identified in laboratory C. pomonella populations, exhibiting excellent biological activity against its host. This virus demonstrates robust biological activity, field control efficacy, thermal stability, and significant UV resistance. Our findings suggest that CypoNPV and its host, C. pomonella , may have co-evolved mechanisms for maintaining mutual stability. Furthermore, our results indicate the feasibility of utilizing insect viruses from stably cultured laboratory populations as potential biocontrol agents and provide a basis for further optimization of their UV stability for efficient pest management. Results Isolation and identification of a novel nucleopolyhedrovirus from laboratory C. pomonella population A nucleopolyhedrovirus was successfully isolated from C. pomonella larvae of LRC population. This isolation occurred in a cohort exhibiting a low apparent infection rate of 3.14%, where a subset of individuals displayed visible signs of viral infection (Fig. 1 A). This isolate was subsequently purified through differential centrifugation and sucrose density gradient centrifugation. Upon inoculation into fourth-instar C. pomonella larvae, the purified isolate induced typical baculovirus symptoms, including body swelling, irregular pink or brown lesions, and postmortem liquefaction, with the cadavers filled with a white, pus-like substance (Fig. 1 B). Ultrastructural analysis confirmed its classification as a nucleopolyhedrovirus. Scanning and transmission electron microscopy (TEM) showed irregular shaped that OBs, predominantly spherical or ellipsoidal, with diameters ranging from 0.5 to 1.1 µm (Fig. 1 C). The occluded virions were rod-shaped, with each OB containing one to six virions (Fig. 1 D). Pairwise genetic distances between this isolate and its closest known relatives, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) and Epinotia granitalis nucleopolyhedrovirus (EpgrNPV), exceeded 0.05 (Table S1 ). These morphological and molecular evidence indicate that this isolate represents a novel nucleopolyhedrovirus infecting the C. pomonella , for which we propose the designation Cydia pomonella nucleopolyhedrovirus (CypoNPV). Whole-genome analysis of CypoNPV The whole-genome sequencing data of CypoNPV have been deposited in the NCBI SRA database under accession number PRJNA1185292. Whole-genome sequencing revealed that the CypoNPV genome spans 112,909 with a G + C content of 34.30%. A total of 124 ORFs exceeding 150 nucleotides were predicted, with minimal overlap between adjacent ORFs (Fig. 2 ). Of these, 113 exhibited homology to previously reported baculovirus ORFs, while 11 were unique to CypoNPV. Based on predicted functions, the 124 ORFs were categorized into six groups: 33 structural protein genes, 10 transcription-related genes, 16 DNA replication-related genes, 11 infection-related genes, 18 auxiliary genes, and 25 genes of unknown function (Table 1 ). Table 1 Functional categories of predicted ORFs in the CypoNPV genome Functional category CypoNPV(ORF Number) Replication (14) Dbp( 14), me53 (16), ie-1 (23), lef11 (27), lef3 (54), lef12 (31), DNA Polymerase (56), helicase (72), dUTPase (84), endonuclease (85), lef2 (104), alkaline exonuclease (110), lef 1 (122), rr1 (124) Transcription (10) lef6 (13), ie-0 (17), 39k (26), p47 (29), lef8 (34), lef9 (45), vlf1 (59), lef4 (67), lef5 (75), pk-1 (3) Structure (34) Polyhedrin (1), p78/83 (2), p10 (10), p49/49k (18), odv-e18 (19), odv-ec27 (20), odv-e66 (32), ac53 (37), vp1054 (40), fp25k (44), desmoplakin (55), ac78 (60), gp41 (61), ac81 (62), tlp20 (63), vp91(64), cg30 (65), vp39 (66), p33 (69), p18 (70), odv-e25 (71), 38k (74), p6.9 (76), p40 (77), p12 (78), p45/p48 (79), p87/vp87 (80), odv-ec43 (82), calyx/pep (89), parg (96), gp16 (101), p24 (102), pkip (105), F-protein (115) Oral infectivity (7) pif5/odv-e56 (9), p74/pif0 (15), pif6 (53), pif4 (73), pif3 (94), pif2 (106), pif1 (114) Auxiliary (21) hoar (4), p26 (11), ubiquitin (25), ADPRase (28), djbp (35), chaB (43), gp37 (46), bro-b (47), chitinase (48), cathepsin (50), iap-2 (51), mtase (52), bro-c (68), p13 (86), iap-1 (88), sod (92), Nrk1 (99), ptp (100), fgf (112), egt (120), 38.7k (123) Unknown (27) Adho30 (12), ac145 (21), ep23/ac146 (22), ac34 (24), Adho3 (30), ac43 (33), ac52 (36), Adho45 (38), Adho44 (39), ASB110/ac55 (41), ac59 (42), ac75 (57), ac76 (58), ac108 (83), ac110 (81), ac117 (90), Adho107 (93), Adho101 (103), ac18 (107), ac19 (108), Adho113 (113), Adho119 (116), Adho120 (117), Adho121 (118), Adho123 (121), ac106 (98), nrdB/rr2 (109) Unique (11) orf5 , orf6 , orf7 , orf8 , orf49 , orf87 , orf91 , orf95 , orf97 , orf111 , orf119 Phylogenetic analysis using 38 baculovirus core genes placed CypoNPV within Alphabaculovirus Group II, indicating a close relationship with CrpeNPV, Adoxophyes honmai nucleopolyhedrovirus, AdhoNPV, and Adoxophyes orana nucleopolyhedrovirus, AdorNPV (Fig. 3 A). Comparative genomic analysis further revealed limited collinearity between CypoNPV and CpGV, with the strongest collinearity observed between CypoNPV and CrpeNPV (Fig. 3 B). This finding was supported by synteny analysis, which demonstrated a high degree of conservation in genome orientation and gene order between CypoNPV and CrpeNPV (Fig. 3 C). Effects of viral infections on population parameters of C. pomonella To investigate if viral infection severity impacts host performance, C. pomonella larvae were reared on a standard diet or a formaldehyde-supplemented diet. Then viral load and key life-table parameters between these groups were compared (Fig. 4 A). The lower viral group (LV) exhibited a 43.40% reduction in viral copy number compared to the normal viral group (NV) (Fig. 4 B). Life-table analysis indicated that this reduction did not enhance population performance; instead, it altered several crucial developmental and reproductive traits. Specifically, the egg period (EP) was significantly shortened by 2.60%, decreasing from 4.62 ± 0.04 d in NV to 4.50 ± 0.04 d in LV. In contrast, the pupal duration (PD) was significantly prolonged by 9.31%, from 9.02 ± 0.16 d to 9.86 ± 0.17 d (Table 2 ). Fecundity (F) was significantly reduced by 30.44%, dropping from 107.73 ± 7.54 to 74.94 ± 5.36 eggs per female (Table 2 ). Table 2 LC 50 of CypoNPV against first-instar larvae of C. pomonella Time (d) LC 50 (OBs/mL) 95% Confidence Interval(OBs/mL) Probit regression equation Correlation coefficient (r) 7 3.1×10 4 1.0×10 4 -1.1×10 5 y = 0.361x-1.627 0.48 14 5.2×10 4 3.1×10 4 -1.8×10 5 y = 0.165x-0.56 0.96 Consistent with these alterations, no significant differences were observed in the intrinsic rate of increase ( r i ) (Fig. 4 C), net reproductive rate ( R₀ ) (Fig. 4 D), or finite rate of increase ( λ ) (Fig. 4 E) between the NV and LV groups. However, the mean generation time ( T ) was significantly extended by 6.28%, increasing from 41.71 ± 0.51 d to 44.33 ± 0.56 d (Fig. 4 F). These results indicate that a decreased viral copy number altered host development and reproductive output without conferring a significant demographic advantage to the colony. Laboratory biological activity of CypoNPV against C. pomonella larvae To ascertain the insecticidal activity of CypoNPV against C. pomonella larvae, we evaluated the larval survival in laboratory conditions subsequent to exposure to a gradient of OB concentrations (Fig. 5 A). CypoNPV exhibited pronounced pathogenicity against first-instar larvae under laboratory conditions. Larval mortality was first observed in the virus-treated groups from 2 days post inoculation (dpi) onwards, while the control group maintained consistent survival throughout the experimental period. A distinct dose-dependent relationship was observed, wherein elevated OB concentrations precipitated an accelerated and more pronounced reduction in larval survival. Specifically, at a concentration of 1.79 × 10 6 OBs/mL, survival decreased rapidly, reaching zero by 7 dpi. Conversely, lower concentrations induced a more gradual yet persistent decline in survival over time (Fig. 5 B). The estimated median lethal concentration (LC 50 ) was 3.1 × 10 4 OBs/mL at 7 dpi and 5.2 × 10 4 OBs/mL at 14 dpi (Table 3 ). Furthermore, the median lethal time (LT 50 ) decreased proportionally with increasing virus concentration, with the lowest LT 50 recorded at 3.18 days at 1.79 × 10 6 OBs/mL (Table 4 ). Table 3 LT 50 of CypoNPV against first-instar larvae of C. pomonella Concentration (OBs/mL) LT 50 (d) 95% Confidence Interval (d) Correlation Coefficient(r) 1.79×10 6 3.18 2.47–3.76 0.77 1.79×10 5 5.08 3.79–6.11 1.00 1.79×10 4 9.53 8.25–11.23 0.99 1.79×10 3 10.20 8.91–12.02 0.99 Table 4 Field control efficacy of CypoNPV against C. pomonella Treatment Fruit damage rate before spraying (%) Fruit damage rate at 7 days after spraying (%) Increase in fruit damage rate (%) Control efficacy (%) Water control 38.33 ± 2a 44.00 ± 2a 5.61 - Lambda-cyhalothrin suspension concentrate 40.60 ± 1.5a 41.00 ± 1b 0.40 86.11 CypoNPV 41.00 ± 3a 42.00 ± 2b 1.00 77.78 Different lowercase letters within the same column indicate significant differences at P < 0.05. Field control efficacy of CypoNPV against C. pomonella populations Field trials indicated that CypoNPV achieved 77.78% control efficacy at 7 days, a level not significantly different from the 86.11% efficacy observed with the chemical insecticide lambda-cyhalothrin (Table 5 ). These results suggest that CypoNPV holds significant potential as a biological control agent against C. pomonella . Table 5 Effects of dietary formaldehyde supplementation on life-history parameters of C. pomonella Parameters NV LV Egg duration (days) 4.62 ± 0.04 4.50 ± 0.04* Larval duration (days) 21.21 ± 0.23 21.33 ± 0.39 Pupal rate (%) 28.67 ± 16.47 27.69 ± 0.04 Pupal duration (days) 9.02 ± 0.16 9.86 ± 0.17* Adult longevity (days) 17.30 ± 0.36 17.58 ± 0,32 Fecundity (eggs/female) 107.73 ± 7.54 74.94 ± 5.36* Total preoviposition period (TPOP) 37.27 ± 0.573 36.06 ± 0.504 Intrinsic rate of increase ( r i , day − 1 ) 0.06 ± 0.0065 0.05 ± 0.0059 Net reproductive rate ( R 0 , eggs) 10.77 ± 2.74 9.22 ± 2.25 Finite rate of increase ( λ , day − 1 ) 1.06 ± 0.0070 1.05 ± 0.0061 Mean generation time ( T , day − 1 ) 41.71 ± 0.51 44.33 ± 0.56* Note: Asterisk (*) indicates a significant difference between the LV and NV groups for the corresponding parameter ( P < 0.05). CypoNPV exhibits excellent thermal stability Thermal pre-treatment of CypoNPV did not significantly diminish its biological activity, as evidenced by the substantial retention of infectivity (Fig. 6 A). Larvae in the water-treated control group exhibited a normal phenotype. In contrast, larvae infected with untreated CypoNPV at 1.79 × 10 5 OBs/mL exhibited a severe phenotype at both 7 and 10 dpi (Fig. 6 B). Pre-treatment at 40°C had minimal impact on viral pathogenicity; severe symptoms persisted, with affected larvae ranging from 83.00% to 93.00% at 7 dpi and 83.00% to 97.00% at 10 dpi, and no normal larvae were observed. Conversely, pathogenicity decreased progressively with increasing pre-treatment temperatures. At 60°C, the incidence of severe symptoms reduced to 7.00%–40.00%, while at 80°C, no severe symptoms were evident, and the majority of larvae remained normal (50.00%-97.00%). Even after pre-treatment at 100°C, residual infectivity was still detectable (Fig. 6 C). Consistent with these phenotypic observations, qPCR analysis showed that viral replication was largely maintained following pre-treatment at 40°C, with viral copy numbers remaining at approximately 85.75%-85.79% of the untreated control at 7 dpi. However, viral replication declined at higher temperatures: 65.59%-73.44% at 60°C, 56.17%-65.35% at 80°C, and 43.08%-48.19% at 100°C (Fig. 6 D). Larval mortality showed a similar trend. Pre-treatment at 40°C resulted in 100.00% mortality retention, whereas mortality retention significantly decreased after pre-treatment at 60°C, 80°C, and 100°C (Fig. 6 E). These results demonstrate that CypoNPV possesses robust thermal stability at 40°C and retains detectable pathogenicity even after exposure to higher temperatures. CypoNPV exhibits considerable UV tolerance CypoNPV maintained detectable biological activity post-UV irradiation (Fig. 7 A). As in the thermal stability assay, all larvae treated with water remained phenotypically normal, whereas larvae infected with CypoNPV at 1.79 × 10 5 OBs/mL exhibited severe phenotypes at both 7 and 10 dpi (Fig. 7 B). Following irradiation at 98 µW/cm², a mixed distribution of symptom classes persevered, with severe symptoms affecting 0.00%-16.70% of larvae at 7 dpi and 0.00%-20.00% at 10 dpi, indicating retention of viral pathogenicity. In contrast, symptom severity declined with increasing irradiation intensity. At 180 and 267 µW/cm², severe symptoms were infrequent or absent, and the proportion of normal larvae increased to 56.70%-86.60% and 76.70%-90.00% at 7 dpi, and 56.70%-80.00% and 50.00%-86.70% at 10 dpi, respectively. At 384 µW/cm², no severe symptoms were observed, with the majority of larvae remained normal (93.30%-96.70% at 7 dpi and 63.30%-90.00% at 10 dpi), although mild or moderate symptoms were still detectable (Fig. 7 C). Consistent with these phenotypic observations, qPCR analysis showed that viral replication remained detectable after UV irradiation, albeit at reduced levels with increasing irradiation intensity. At 7 dpi, viral copy numbers were 61.22%-69.76% of the untreated control following irradiation at 98 µW/cm², compared to 61.29%-66.29% at 180 µW/cm², 61.01%-65.33% at 267 µW/cm², and 49.20%-61.75% at 384 µW/cm² (Fig. 7 D). Larval mortality showed a similar trend. At 7 dpi, the retention of mortality ranged from 13.33%-53.33% at 98 µW/cm², 13.33%-46.67% at 180 µW/cm², 10.00%-43.33% at 267 µW/cm², and 6.67%-16.67% at 384 µW/cm². By 10 dpi, the corresponding ranges were 26.67%-86.67%, 20.00%–-65.00%, 13.33%-50.00%, and 10.00%-36.67%, respectively (Fig. 7 E). These results indicate that CypoNPV retains significant biological activity post-UV exposure, particularly at lower irradiation intensities, demonstrating considerable UV tolerance. Discussion Typically, baculoviruses developed for biological control are more frequently identified from naturally infected field populations or widespread outbreaks rather than from indoor colonies. Although this observation appears atypical, covert baculovirus infections are known to occur regularly in both natural and laboratory insect populations, potentially persisting for extended durations without apparent disease manifestations 2 , 15 , 26 , 27 . Consequently, the discovery of covertly infected viral strains within a laboratory colony that exhibit pathogenicity after propagation presents a novel avenue for pest biological control. This possibility aligns with growing evidence indicating that agriculturally significant insects harbor diverse and biologically active viromes 28 , 29 . Some of these viruses exhibit host-specific distributions, while others maintain cross-host infectivity, reflecting long-term coevolutionary structuring of viral assemblages 28 . In this study, we identified a novel nucleopolyhedrovirus CypoNPV from a stably maintained laboratory population of C. pomonella . The significant genetic proximity between CypoNPV and CrpeNPV is noteworthy, particularly given that CrpeNPV was initially isolated from a laboratory-reared C. peltastica population and subsequently demonstrated efficient infectivity toward C. pomonella . This observation suggests that baculoviruses associated with tortricid hosts can maintain infectivity across related species 19 , 20 . From an evolutionary perspective, this aligns with the prevailing understanding that while baculoviruses generally exhibit restricted host ranges, host utilization is often conserved among closely related insect species and can be punctuated by host shifts between them 30 . Consequently, the close relationship between CypoNPV and CrpeNPV could stem from either divergence from a recent common ancestor within a tortricid-associated alphabaculovirus lineage or host-associated speciation following adaptation to distinct, yet phylogenetically related, tortricid hosts 31 . Based on this evidence, it is reasonable to hypothesize that CypoNPV might also retain pathogenicity against C. peltastica or other closely related tortricids. However, this remains an inference derived from phylogenetic relatedness and the established cross-infectivity of CrpeNPV, necessitating direct validation through cross-infection bioassays. These findings highlight the importance of considering baculovirus evolution in laboratory colonies not only in terms of persistence but also in relation to long-term host association, tortricid-specific diversification, and potential host shifts among closely related species. The isolated CypoNPV, after being multiplied, showed excellent biologically activity and orchard control efficacy against C. pomonella . While direct LC 50 comparison across baculovirus systems require caution due to variations in host species, larval instar, and bioassay methodology, the rapid kill observed with CypoNPV is comparable to the general efficacy of CpGV, which typically results in susceptible larvae succumbing within 3–7 dpi 7 . CypoNPV does not represent the fastest-acting baculoviruses, as certain S. frugiperda multiple nucleopolyhedrovirus (SfMNPV) isolates can induce larval mortality under 56 h, but its virulence aligns with that of lepidopteran baculoviruses considered promising candidates for development 32 . Notably, the orchard efficacy of CypoNPV (77.78%) falls within the established operational range of CpGV products (75–90%) for C. pomonella management 7 . This performance is also consistent with other insect viruses deemed worthy of development, a co-occluded HearNPV mixture caused 89–100% larval mortality on treated tomato plants and performed comparably to Bacillus thuringiensis and spinosad in reducing fruit damage 33 . Likewise, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) showed high efficacy in greenhouse sweet pepper crops, where virus-induced mortality of larvae collected from treated plants reached 70–89%, and recent feeding damage in heavily infested greenhouses declined from 45–94% before treatment to 0.1–9.9% after two applications 34 . Importantly, when evaluated against the lambda-cyhalothrin positive control in the present orchard trial, CypoNPV still provided agriculturally significant population suppression, demonstrating its efficacy not only in laboratory settings but also under orchard conditions. This is particularly relevant given the increasing challenges in C. pomonella management, including the limited availability of registered insecticides 35 , the growing of insecticide resistance cases to lambda-cyhalothrin in field populations 36 , 37 . Furthermore, current commercial virus products for C. pomonella control are predominantly based on CpGV formulations 38 , 39 . These findings indicate that CypoNPV integrates biologically significant virulence with practical field effectiveness, positioning it as a valuable novel baculoviral resource for codling moth management, especially considering the limited dedicated control options and escalating resistance to conventional insecticides. The development and field application of insect-pathogenic viruses depend not only on pathogenicity but also on their thermal sensitivity, and UV sensitivity 24 . Temperature effects are best understood by considering the microclimate on exposed fruit surfaces where the virus is deposited, rather than solely ambient air temperature. C. pomonella typically oviposits on fruit surfaces, with newly hatched larvae seeking a suitable location before boring into the fruit. Fruit surface temperatures can significantly exceed air temperature, potentially reaching 15℃ above the daily maximum air temperature under clear summer conditions (Konno et al., 2024). Sunburn browning under sunlight exposure has been linked to fruit surface temperatures between approximately 46–49℃ 40 . In this study, we found complete retention of CypoNPV activity after a 4-h pre-treatment at 40℃, indicating that CypoNPV can withstand the thermal load that experienced by sun-exposed fruit surfaces throughout the season. Furthermore, the observed decline at 60–100℃ likely signifies the upper thermal stability limit of the virus, rather than conditions typically encountered in the orchards. Nevertheless, this limit remains relevant for practical applications, as transient heat stress can occur during spray preparation, transport, storage, and on highly exposed plant surfaces post-application. Even after pre-treatment at 100°C, mild symptoms were still observed in a small proportion of larvae, indicating that the virus retained detectable residual infectivity under extreme heat exposure. These findings suggest CypoNPV possesses strong thermal robustness and retains substantial biological activity, further supporting its promise as an effective candidate for practical control. Survey shows that monthly mean UV intensities from April to September 2025 in 15 C. pomonella occurrence regions across China ranged from 339.93 to 398.36 µW/cm 2 (WheatA agricultural meteorological big data platform: https://wheata.cn/ , version 1.6.9; accessed on 9 April 2026). In Gaizhou City, Liaoning Province, the field efficacy trial location for this study, the corresponding values ranged from 341.82 to 371.08 µW/cm 2 (Table S2). Consequently, the 384 µW/cm 2 treatments employed in this study align with or approach the upper range of the mean seasonal UV levels documented across C. pomonella regions, thus representing realistic exposure conditions. Conversely, the lower treatments of 98 and 180 µW/cm 2 can be interpreted as reduced-exposure scenarios, also pertinent to orchard environments, as virus deposits are not constantly subjected to full solar radiation post-application. Against this background, the preservation of measurable infectivity after pretreatment at 98 and 180 µW/cm 2 suggests that CypoNPV can retain activity under lower or partially shielded exposure conditions. In contrast, the observed progressive reduction in activity at 267 and 384 µW/cm 2 indicates that solar radiation likely imposes a more significant limitation on field persistence than temperatures. However, even after UV irradiation, a considerable proportion of larvae still exhibited mild and moderate symptoms, suggesting that CypoNPV retained biologically relevant infectivity and could still cause progressive disease in treated larvae. This pattern is not atypical for baculoviruses. Within C. pomonella management systems, the limited residual activity of CpGV under orchard conditions has long been recognized as a practical constraint, necessitating frequent reapplication at 7- to 10-d intervals due to rapid solar exposure-induced reduction in persistence on fruit and foliage 41 . These findings suggest that the UV sensitivity of CypoNPV should primarily be addressed as a formulation and application challenge, with future enhancements most likely stemming from the development of solar-protective formulations, canopy-targeted deposition strategies, and optimized spray timing to minimize cumulative UV exposure. Formaldehyde has historically served as an antiviral or prophylactic agent in insect rearing, particularly against baculoviruses like NPV in lepidopteran colonies 42 . To ascertain the impact of CypoNPV infection on population development, we administered formaldehyde through the diet, aiming to eradicate or reduce viral titers. Our findings indicated that while viral copy number decreased by 43.40%, there was no corresponding enhancement in demographic performance; instead, mean generation time increased. This challenges the prevailing assumption that persistent viral load is the primary constraint on host population growth 15 . Similar phenomena have been observed in other insect-virus systems; for instance, ribavirin treatment in Mediterranean fruit flies reduced RNA virus levels without improving host fitness 43 , and experimental suppression of dengue virus in Aedes aegypti similarly failed to confer fitness benefits 44 . These results suggest a non-linear relationship between viral burden and host demographic performance. In our study, reducing CypoNPV copy number did not confer a measurable colony-level advantage, reinforcing the conclusion that CypoNPV load was not the principal driver of population development in this laboratory colony. This aligns with broader evidence indicating that latent or persistent insect virus infections do not invariably yield uniform or predictable fitness outcomes 45 – 47 . Reciprocal host-virus selection balances persistence, transmission, and host survival, not necessarily maximal virulence 1 , 16 . Persistent and heritable viruses play a critical role in insect ecology and evolution, moving beyond the notion of incidental infections with minor biological impact 47 , 48 . This broadened perspective is reinforced by recent virome studies, which demonstrate that viral prevalence, abundance, and diversity are modulated by ecological and evolutionary filters, rather than purely stochastic processes 49 . Moreover, studies on naturally occurring viral infections in laboratory insect populations have shown that persistent viruses can influence host lifespan and reproductive fitness, even in the absence of overt epidemics, suggesting that enduring viral associations retain biological significance under laboratory rearing conditions 50 . Collectively, the identification of CypoNPV within an indoor population implies that laboratory colonies can harbor latent virus diversity without compromising it biological relevance (Fig. 8 ). These insights advance our comprehension of baculoviruses in insects and provide a more robust framework for integrating viral persistence, coevolution, environmental stability, and the practical deployment of insect viruses developed in laboratory settings. From a practical viewpoint, this study offers novel biological control agents for effective C. pomonella management and introduces innovative strategies to combat the growing challenge of insecticide resistance. Methods Insects and Virus A laboratory-reared colony of C. pomonella (LRC) was maintained in a Panasonic incubator ( MLR-352H-PC, Japan) under controlled conditions: 26 ± 1°C temperature, 60 ± 5% relative humidity, and a 16:8 hours (light: dark) photoperiod 51 . Larvae exhibiting typical symptoms of baculovirus infection were collected and immediately stored at -80°C for subsequent analysis. To isolate occlusion bodies (OBs), a crude extract was prepared from symptomatic larvae, followed by OBs purification using 50–60% sucrose gradient centrifugation, as previously detailed 52 . Electron Microscopy For transmission electron microscope (TEM) examination, purified OBs were placed on carbon-coated grids and negatively stained with 1% aqueous uranyl acetate (w/v), as described previously 53 . The grids were then examined using a Hitachi HT7700 TEM (Hitachi, Tokyo, Japan) at an 80 kV accelerating voltage. For cross-sectional morphological analysis, OBs were embedded in 1% agarose and fixed in 1% osmium tetroxide for 2 h at room temperature. Following fixation, samples were rinsed, dehydrated, embedded, and sectioned. Ultrathin sections (80 nm thick) were prepared using a Leica UC7 ultramicrotome, collected onto 150-mesh copper grids, and double-stained with 2% uranyl acetate and lead citrate 54 . Finally, the grids were imaged with the Hitachi HT7700 TEM at an accelerating voltage of 100 kV. Gene amplification and complete genome sequencing Total genomic DNA was extracted from insect samples using the E.Z.N.A.® Viral DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's protocols. Subsequently, partial regions of the polh / gran , lef-8 , and lef-9 genes were amplified using degenerate primer pairs (prPH-1/prPH-2, prL8-1/prL8-2, prL9-1/prL9-2), as previously described 55 Whole-genome sequencing was performed by Sangon Biotech (Shanghai, China) on the Illumina HiSeq platform, generating a total of 47,391,588 raw reads. Quality control and adapter trimming were conducted using Trimmomatic (v0.36) to obtain high-quality clean reads. De novo assembly was performed with SPAdes (v3.5.0). The service provider initially annotated the resulting genome using Prokka (v1.1.0). Open reading frames (ORFs) encoding peptides of ≥ 50 amino acids were predicted with ORF Finder. Annotation accuracy was enhanced through a refinement process using closely related nucleopolyhedrovirus genomes, specifically CrpeNPV (accession no. MH394321), as references. Phylogeny and Kimura 2-parameter analysis To evaluate genetic relationships, protein sequences encoded by CypoNPV were compared with their homologs in reference genomes using BioEdit (v7.0.9.0) with default settings. Multiple genome alignments were conducted using the progressive Mauve algorithm in Geneious v11.0.2 56 . Gene parity plots were then generated to visualize syntenic relationships and organizational conservation between CypoNPV and other baculoviruses, following established methods 57 . To infer phylogenetic relationships among baculoviruses, protein sequences of 38 core genes were extracted from 107 complete baculovirus genomes and aligned using MAFFT under the auto strategy and normal alignment mode. Poorly aligned regions were subsequently removed using Gblocks with default parameters. The aligned protein sequences of the 38 core genes were concatenated into a supermatrix using PhyloSuite (version 1.2.2). The optimal partitioning scheme for maximum-likelihood (ML) analysis was determined using PartitionFinder2, based on the Bayesian information criterion (BIC) and employing a greedy search algorithm with linked branch lengths. Finally, an ML tree was constructed with IQ-TREE, utilizing ultrafast bootstrap approximation with 5000 replicates and the SH-aLRT test with 1000 replicates. For genetic divergence analysis, pairwise Kimura 2-parameter (K2P) distances were calculated from aligned nucleotide sequences of polh , lef-8 , lef-9 , the concatenated polh / lef-8 / lef-9 fragment, and the concatenated 38 core genes using MEGA (version 10.1.8) 58 . Site variation was modeled as uniform, and gaps were treated as pairwise deletions. Species demarcation was conducted using adjusted K2P thresholds: for single or concatenated polh / lef-8 / lef-9 genes, thresholds were set as K2P 0.050 (different species). In contrast, for the concatenated 38 core-gene fragments, the corresponding thresholds were K2P 0.072 (different species), following criteria established previously 59 . Analysis of viral copy numbers Total viral DNA was extracted from the larvae using the EasyPure Viral DNA/RNA Kit. Samples were processed in groups of 3–4 individuals, with three replicates per condition. The extracted DNA was eluted in 30 µL of sterile ddH₂O. The polyhedrin gene, a conserved core gene of CypoNPV, was selected as the target for quantifying viral replication due to its consistent correlation with viral genomic copies. A quantitative standard was constructed by amplifying the polyhedrin gene via PCR, followed by gel extraction, cloning into a pMD™-19 vector, and transformation. Positive clones (designated T-polh) were verified by sequencing, cultured, and subjected to plasmid extraction. The plasmid concentration (ng/µL) was measured for copy number calculation. Viral copy number was calculated using the following formula: Population parameters of infected with different concentrations of viruses For life-table analysis, newly laid eggs from the laboratory colony were divided into two groups: the normal viral group (NV), fed a standard diet, and the lower viral group (LV), fed a formaldehyde-supplemented diet. In the LV group, formaldehyde was incorporated into the artificial diet at approximately three times the original formulation's level, specifically 200 µL formaldehyde per 50 mL diet. This concentration was determined from preliminary experiments, as it effectively reduced viral copy number without significantly impacting survival rates. Three replicates of 15 eggs each were randomly assigned to each treatment and maintained under identical environmental conditions. Individuals were monitored daily to record the developmental duration of each life stage and their survival status. Upon hatching, larvae were provided with an artificial diet, which was replaced every 5 days until pupation. Pupal duration and adult emergence were recorded, and newly emerged adults were paired and kept under the same rearing conditions for oviposition. Fecundity and adult survival were documented daily until all individuals had expired. Data analysis was carried out Population parameters, including the intrinsic rate of increase ( r i ), finite rate of increase ( λ ), net reproductive rate ( R 0 ), and mean generation time ( T ), were calculated using the TWOSEX-MSChart software and bootstrap methods for standard error estimation 60 , 61 . Laboratory biological activity of CypoNPV To evaluate the biological activity of purified CypoNPV OBs against neonate larvae, we employed a droplet-feeding method (Fig. 5 A). OB concentrations were determined using a hemocytometer (Thoma cell counting chamber), and a six-point, ten-fold serial dilution series was prepared, resulting in doses ranging from 1.79 × 10⁶ to 1.79 × 10¹ OBs/mL. For each concentration, 2 µL of OB suspension was applied to the surface of an artificial diet cube (0.5 × 0.5 × 2 cm). After air-drying, the diet blocks were placed into individual wells of a 24-well plate. Newly hatched larvae (< 8 h old) were introduced to the treated diet and kept under standard rearing conditions. After 24 h, larvae were transferred to fresh, untreated diet, and any deceased individuals were removed. Each dose was tested on 30 neonate larvae, with sterile water serving as the negative control. The entire bioassay was repeated three times independently. Plates were incubated at 26 ± 1°C and 60 ± 5% relative humidity, and larval mortality was recorded daily for 15 days. Field control efficacy of CypoNPV To further evaluate CypoNPV insecticidal efficacy under field conditions, we conducted a trial in an apple orchard located in Wumeifang Village, Jiuzhai Town, Gaizhou City, Liaoning Province, China (40°3’5’’ N, 122°5’44’’ E) from August 14 to August 22, 2024. Before treatment, we assessed the number of infested fruits to establish a baseline. CypoNPV was applied at a concentration of 1.79 × 10⁵ OBs/mL using a motorized sprayer during evening hours, ensuring even coverage on the leaf surface until runoff. Each treatment, including the untreated control, consisted of six apple trees, with a total of 12 L of spray solution applied per treatment. Alongside the viral treatment, we included a negative control (water) and a positive control consisting of 25 g/L lambda-cyhalothrin emulsifiable concentrate (EC) (Syngenta, China), diluted 3000-fold with water and applied using a backpack sprayer. Post application, fruit damage was evaluated by examining over 25 fruits per tree from each of the four cardinal directions (east, south, west, and north). Field control efficacy was calculated at 7 days post-application, according to the Chinese national standard GB/T 17980.65–2004. Thermal and ultraviolet pretreatment of CypoNPV Purified CypoNPV OB suspensions were adjusted to 1.79 × 10 5 OBs/mL and used for thermal and ultraviolet (UV) stability assays. For the thermal treatment, aliquots of the viral suspension were incubated in a metal bath at 40, 60, 80, or 100°C for 1, 2, 3, or 4 h, respectively (Fig. 6 A). For the UV treatment, viral suspensions at the same concentration were exposed to UV intensities of 98, 180, 267, or 384 µW/cm² for 1, 2, 3, or 4 h (Fig. 7 A). To minimize the shielding effect of the water layer, 0.3 mL of viral suspension was first dispensed into a Petri dish and air-dried at room temperature in the dark before irradiation. The untreated viral suspension served as the control group. Subsequently, the treated virus was then subjected to the same third-instar feeding bioassay described above, with 20 larvae per treatment and three independent replicates, as third-instar larvae provided enhanced visibility and differentiation of symptom progression Infection symptoms and larval mortality were recorded at 7 and 10 days post-inoculation. For each time point, 9–12 larvae were collected, weighed, and stored at -20°C for subsequent viral DNA extraction. This weighing enabled later normalization of viral copy number per unit body weight. Data analysis The log-probit regression equation and LC 50 (and LT 50 ) value were determined by probit analysis using SPSS 26.0. The results were graphically represented using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA), with all values being expressed as mean ± standard error. Furthermore, significant difference in field efficacy between CypoNPV and control was analyzed through independent samples t -tests using SPSS 26.0. Declarations Acknowledgements This research was supported by the Liaoning Distinguished Youth Scholars Science Foundation (2024JH3/50100027), National Key Research and Development Program (2021YFD1400201), and the Education Department of Liaoning Province (JYTYB2024038). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9410995","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624587606,"identity":"97fd1230-e232-4456-80f4-c2b41454fa7b","order_by":0,"name":"Yuxi Liu","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuxi","middleName":"","lastName":"Liu","suffix":""},{"id":624587608,"identity":"f7caa1f2-d45e-47f3-b9d7-7c4f624e5dfe","order_by":1,"name":"Huanjuan Zhao","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huanjuan","middleName":"","lastName":"Zhao","suffix":""},{"id":624587610,"identity":"99eade71-129a-4ae2-bdf5-2b882a0cb290","order_by":2,"name":"Bokun Wang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bokun","middleName":"","lastName":"Wang","suffix":""},{"id":624587614,"identity":"48079d80-5537-46e4-b77c-dc1c55181cf2","order_by":3,"name":"Yu Fei","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Fei","suffix":""},{"id":624587615,"identity":"e811e4ed-9707-4d04-beb3-565233eb9eac","order_by":4,"name":"Bing Bai","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Bai","suffix":""},{"id":624587621,"identity":"13ecbd95-d21e-425e-80a0-84bfc975d15d","order_by":5,"name":"Ping Gao","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Gao","suffix":""},{"id":624587622,"identity":"828a4e3b-b251-4c2b-99b5-5f29dfc1c7ca","order_by":6,"name":"Yuting Li","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Li","suffix":""},{"id":624587625,"identity":"6eacd315-f7b6-44d3-9e19-3955122d1cf4","order_by":7,"name":"Xueqing Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYPACGyjNRryWNNK1HCZBi8HxHMPPBb/O2xscP2PA8KHsMAP/7Ab8WiR73hhLz+y7nbjhTI4B44xzhxkk7hzAr4VfIsdAmrfndoLBgRwDZt62wwwGEgn4tbBJ5Bj/5u05Z29w/o0B819itABtMZPm+XGAccMNoC2MxGiR7HlWZs3bkJw488azgoM959J5JG4Q0GJwPHnzbZ4/dvZ855M3PvhRZi3HP4OAFgaGDAMGxjYGBoUDDAxAxMBDSD0QpD9gYPjDwCDfQITaUTAKRsEoGJkAADCqRUt+gjBtAAAAAElFTkSuQmCC","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xueqing","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-04-14 05:58:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9410995/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9410995/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107503732,"identity":"34bfb5cc-de6e-4dcb-9e21-6e4d5db56743","added_by":"auto","created_at":"2026-04-22 06:27:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5754229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characteristics of CypoNPV-infected larvae and purified virus particles. \u003c/strong\u003eA diseased \u003cem\u003eC. pomonella\u003c/em\u003e larva collected from the laboratory colony (A). Healthy and virus-infected larvae after inoculation with purified CypoNPV (B). Purified occlusion bodies observed by scanning electron microscopy (C). A single occlusion body containing rod-shaped virions observed by transmission electron microscopy (D).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/8f3f437a8bae5887fa4a0e6b.png"},{"id":107705912,"identity":"b8577948-56c5-4d13-8365-f6b17a7f6842","added_by":"auto","created_at":"2026-04-24 09:15:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1678758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircular genome map of CypoNPV. \u003c/strong\u003ePredicted open reading frames on both strands of the CypoNPV genome, with arrow direction indicating transcriptional orientation. ORFs are color-coded as baculovirus core genes, lepidopteran conserved genes, other baculoviral genes, and unique ORFs.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/8e3ba82647eb9261cc735616.jpg"},{"id":107705272,"identity":"a8d2c47e-6259-49f9-8cfa-c14476d5b73c","added_by":"auto","created_at":"2026-04-24 09:10:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2874607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic position and comparative genomic analysis of CypoNPV. \u003c/strong\u003ePhylogenetic tree based on 38 baculovirus core genes (A). Gene parity plots comparing CypoNPV with representative baculoviruses (B). Collinearity analysis among CypoNPV, CrpeNPV, AdhoNPV, and AdorNPV (C).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/30d069f942c577514a3ef204.jpg"},{"id":107503738,"identity":"5c66f510-3272-4709-9e5e-ac0b8fb4115a","added_by":"auto","created_at":"2026-04-22 06:27:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1335368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of covert viral load and population parameters in normal- and low-viral-load populations of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eIndividuals were reared on either a standard artificial diet (NV) or a formaldehyde-supplemented diet (LV), after which viral load and life-table traits were evaluated. Schematic illustration of the experimental design (A). Viral copy number, expressed as log10 copies mg⁻¹ body weight, was quantified by qPCR (B). Life-table parameters, including the intrinsic rate of increase (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e), net reproductive rate (\u003cem\u003eR₀\u003c/em\u003e), finite rate of increase (\u003cem\u003eλ\u003c/em\u003e), and mean generation time (\u003cem\u003eT\u003c/em\u003e), were compared between the two treatments (C-F). Bars represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/0d8685e5f042702cff3f1f6f.jpg"},{"id":107503735,"identity":"187f8f89-169d-4579-941c-a034a69b1e23","added_by":"auto","created_at":"2026-04-22 06:27:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1001051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival of first-instar \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e larvae after exposure to different concentrations of CypoNPV. \u003c/strong\u003eSchematic illustration of the experimental design (A). Survival curves of larvae treated with different concentrations of CypoNPV (B). The untreated group was used as the control.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/93541f01d32ff7da69a3c035.jpg"},{"id":107705931,"identity":"f14ef236-ef3b-4407-b11f-69c351159386","added_by":"auto","created_at":"2026-04-24 09:15:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1637756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermal stability of CypoNPV. \u003c/strong\u003eSchematic illustration of the heat pretreatment applied to purified CypoNPV occlusion bodies before inoculation of \u003cem\u003eC. pomonella\u003c/em\u003e larvae, with treatments at 40, 60, 80, or 100°C for 1, 2, 3, or 4 h (A). Representative larval symptom phenotypes classified as normal, mild, moderate, and severe (scale bar = 5 mm) (B). Proportions of symptom phenotypes at 7 and 10 dpi in larvae inoculated with heat-pretreated virus; water and untreated virus served as the negative and positive controls, respectively (C). Viral copy number in infected larvae, quantified by qPCR and expressed as log10 copies mg⁻¹ body weight, at 1, 3, 5, and 7 dpi after 1 h or 3 h heat pretreatment (D). Larval mortality at 7 and 10 dpi following heat pretreatment for 1-4 h at the indicated temperatures (E). Data are presented as mean ± SEM, and different lowercase letters indicate significant differences among treatments within the same sampling time or treatment duration (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/5c322c2acf259c4d8af1bd01.jpg"},{"id":107705289,"identity":"5b77d5d5-1d0b-4421-a991-b0d46fbb3139","added_by":"auto","created_at":"2026-04-24 09:10:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1679388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltraviolet stability of CypoNPV. \u003c/strong\u003eSchematic illustration of the UV irradiation treatment applied to purified CypoNPV occlusion bodies before inoculation of \u003cem\u003eC. pomonella\u003c/em\u003elarvae, with irradiation intensities of 98, 180, 267, or 384 μW/cm² for 1, 2, 3, or 4 h (A). Representative larval symptom phenotypes classified as normal, mild, moderate, and severe (scale bar = 5 mm) (B). Proportions of symptom phenotypes at 7 and 10 dpi in larvae inoculated with UV-irradiated virus; water and untreated virus served as the negative and positive controls, respectively (C). Viral copy number in infected larvae, quantified by qPCR and expressed as log10 copies mg⁻¹ body weight, at 1, 3, 5, and 7 dpi after 1 h or 3 h UV irradiation (D). Larval mortality at 7 and 10 dpi following UV irradiation for 1-4 h at the indicated intensities (E). Data are presented as mean ± SEM, and different lowercase letters indicate significant differences among treatments within the same sampling time or treatment duration (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/32413f68fea18290422958e7.jpg"},{"id":107705559,"identity":"a214302c-077c-4d66-b3ce-920f77190764","added_by":"auto","created_at":"2026-04-24 09:13:31","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2326862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical summary of the isolation and biological characteristics of CypoNPV from a laboratory-reared \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e population. \u003c/strong\u003eVirus-like infection symptoms were observed in the laboratory colony, leading to the isolation of CypoNPV. The isolate remained pathogenic to \u003cem\u003eC. pomonella\u003c/em\u003e and exhibited favorable thermal stability and UV tolerance, indicating its potential for biological control applications.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/99baa0267ec544b304ff7e16.jpg"},{"id":107709061,"identity":"ba3addd4-ffbc-416e-8a60-f75a7809bda8","added_by":"auto","created_at":"2026-04-24 09:34:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19174659,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/80286a53-bc54-417c-a5da-05c6ae0d17da.pdf"},{"id":107705643,"identity":"4cd125d8-0b07-4eb8-975e-4f9cb50f8d2c","added_by":"auto","created_at":"2026-04-24 09:14:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19300,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-9410995/v1/d37e4c034c1d90feba825491.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A new nucleopolyhedrovirus isolated from a laboratory population of Cydia pomonella exhibits excellent biological activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVirus-host interactions represent an evolutionary arms race, where reciprocal selection pressures for pathogenicity and resistance dictate ecological outcomes, ranging from mutualism to lethality \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Insect-pathogenic viruses are ubiquitous in natural insect populations, with baculoviruses being the most extensively studied due to their high host specificity, potent pathogenicity, and long-standing significance in biological control \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This association has been recognized for over a century: polyhedrosis in silkworm was documented in the mid-19th century, and the silkworm-baculovirus system subsequently became a well-established model for insect-virus interactions \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In orchards, \u003cem\u003eCydia pomonella\u003c/em\u003e granulovirus (CpGV) serves as a benchmark, identified from naturally infected codling moth larvae and developed into highly effective products for pest management \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eAnticarsia gemmatalis\u003c/em\u003e multiple nucleopolyhedrovirus (AgMNPV) was widely deployed against the velvetbean caterpillar in Brazilian soybeans, regarded as one of the most successful viral bioinsecticides developed \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Commercial applications of heliothine baculoviruses further underscore the practical value of insect viruses, \u003cem\u003eHelicoverpa zea\u003c/em\u003e SNPV formed the basis of Elcar, and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e SNPV/HearNPV became a key commercial insecticide for bollworm control in China \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Their utility extends beyond annual crops, with \u003cem\u003eLymantria dispar\u003c/em\u003e multiple nucleopolyhedrovirus (LdMNPV) developed as Gypchek for operational management in forest ecosystems \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. More recently, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e multiple nucleopolyhedrovirus (SfMNPV) has been applied to management, with registered products like Cartugen and Fawligen, and ongoing research supporting its biological activity and practical potential \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These cases collectively demonstrate the proven biological activity and, in several instances, the clear practical and commercial value of insect pathogenic viruses.\u003c/p\u003e \u003cp\u003eViruses detected in laboratory colonies, rather than as overt disease outbreaks, often exist as covert, persistent, low-level, or asymptomatic infections \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These infections can remain undetected for extended periods, becoming apparent only under specific physiological or environmental conditions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, viral infections do not negate the biological relevance of laboratory populations. On the contrary, baculovirus covert infections are prevalent in laboratory insect populations, and can be maintained through various transmission routes \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The hidden persistence of these viruses suggest that indoor insect colonies may represent an overlooked source of candidate viruses for future biological control. This potential is not merely theoretical; a covert infection of \u003cem\u003ePhthorimaea operculella\u003c/em\u003e granulovirus in a laboratory colony demonstrated stable persistence and influenced host population dynamics \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Latent SpltNPV was also identified in apparently healthy laboratory \u003cem\u003eSpodoptera litura\u003c/em\u003e population and could be reactivated following heterologous virus challenge \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Similarly, a novel alphabaculovirus isolated from a laboratory-reared \u003cem\u003eCryptophlebia peltastica\u003c/em\u003e population exhibited virulence against its native host and was proposed as a potential biopesticide; and a subsequent study confirmed it high infectivity to \u003cem\u003eC. pomonella\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Even in the case of \u003cem\u003eC. pomonella\u003c/em\u003e, a granulovirus from a British Columbia laboratory colony was found to be identical or nearly identical to orchard isolates \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These findings imply that long-term laboratory insect colonies may serve as an underexplored reservoir for candidate insect viruses.\u003c/p\u003e \u003cp\u003eWhile pathogenicity is a prerequisite, it alone does not suffice to confirm the applied value of these viruses \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Solar ultraviolet (UV) radiation rapidly degrades the efficacy of baculovirus on exposed surfaces, and temperature exerts a significant influence on viral activity, production, and persistence \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, a thorough evaluation of thermal stability, in conjunction with UV tolerance, is essential for assessing the realistic field potential of a laboratory-derived isolate \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it remains largely unclear whether viruses persisting in laboratory colonies can be isolated as biologically active agents and whether they retain sufficient pathogenicity and environmental stability for practical application.\u003c/p\u003e \u003cp\u003eThis study presents the first report in insects of a novel nucleopolyhedrovirus (CypoNPV) identified in laboratory \u003cem\u003eC. pomonella\u003c/em\u003e populations, exhibiting excellent biological activity against its host. This virus demonstrates robust biological activity, field control efficacy, thermal stability, and significant UV resistance. Our findings suggest that CypoNPV and its host, \u003cem\u003eC. pomonella\u003c/em\u003e, may have co-evolved mechanisms for maintaining mutual stability. Furthermore, our results indicate the feasibility of utilizing insect viruses from stably cultured laboratory populations as potential biocontrol agents and provide a basis for further optimization of their UV stability for efficient pest management.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIsolation and identification of a novel nucleopolyhedrovirus from laboratory\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e \u003cb\u003epopulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA nucleopolyhedrovirus was successfully isolated from \u003cem\u003eC. pomonella\u003c/em\u003e larvae of LRC population. This isolation occurred in a cohort exhibiting a low apparent infection rate of 3.14%, where a subset of individuals displayed visible signs of viral infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This isolate was subsequently purified through differential centrifugation and sucrose density gradient centrifugation. Upon inoculation into fourth-instar \u003cem\u003eC. pomonella\u003c/em\u003e larvae, the purified isolate induced typical baculovirus symptoms, including body swelling, irregular pink or brown lesions, and postmortem liquefaction, with the cadavers filled with a white, pus-like substance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Ultrastructural analysis confirmed its classification as a nucleopolyhedrovirus. Scanning and transmission electron microscopy (TEM) showed irregular shaped that OBs, predominantly spherical or ellipsoidal, with diameters ranging from 0.5 to 1.1 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The occluded virions were rod-shaped, with each OB containing one to six virions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePairwise genetic distances between this isolate and its closest known relatives, \u003cem\u003eCryptophlebia peltastica\u003c/em\u003e nucleopolyhedrovirus (CrpeNPV) and \u003cem\u003eEpinotia granitalis\u003c/em\u003e nucleopolyhedrovirus (EpgrNPV), exceeded 0.05 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These morphological and molecular evidence indicate that this isolate represents a novel nucleopolyhedrovirus infecting the \u003cem\u003eC. pomonella\u003c/em\u003e, for which we propose the designation \u003cem\u003eCydia pomonella\u003c/em\u003e nucleopolyhedrovirus (CypoNPV).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eWhole-genome analysis of CypoNPV\u003c/h2\u003e \u003cp\u003eThe whole-genome sequencing data of CypoNPV have been deposited in the NCBI SRA database under accession number PRJNA1185292. Whole-genome sequencing revealed that the CypoNPV genome spans 112,909 with a G\u0026thinsp;+\u0026thinsp;C content of 34.30%. A total of 124 ORFs exceeding 150 nucleotides were predicted, with minimal overlap between adjacent ORFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of these, 113 exhibited homology to previously reported baculovirus ORFs, while 11 were unique to CypoNPV. Based on predicted functions, the 124 ORFs were categorized into six groups: 33 structural protein genes, 10 transcription-related genes, 16 DNA replication-related genes, 11 infection-related genes, 18 auxiliary genes, and 25 genes of unknown function (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFunctional categories of predicted ORFs in the CypoNPV genome\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFunctional category\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCypoNPV(ORF Number)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReplication (14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDbp(\u003c/em\u003e14), \u003cem\u003eme53\u003c/em\u003e(16), \u003cem\u003eie-1\u003c/em\u003e(23), \u003cem\u003elef11\u003c/em\u003e(27), \u003cem\u003elef3\u003c/em\u003e(54), \u003cem\u003elef12\u003c/em\u003e(31), \u003cem\u003eDNA Polymerase\u003c/em\u003e(56), \u003cem\u003ehelicase\u003c/em\u003e(72), \u003cem\u003edUTPase\u003c/em\u003e(84), \u003cem\u003eendonuclease\u003c/em\u003e(85), \u003cem\u003elef2\u003c/em\u003e(104), \u003cem\u003ealkaline exonuclease\u003c/em\u003e(110), lef\u003cem\u003e1\u003c/em\u003e(122), \u003cem\u003err1\u003c/em\u003e(124)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTranscription (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003elef6\u003c/em\u003e(13), \u003cem\u003eie-0\u003c/em\u003e(17), \u003cem\u003e39k\u003c/em\u003e(26), \u003cem\u003ep47\u003c/em\u003e(29), \u003cem\u003elef8\u003c/em\u003e(34), \u003cem\u003elef9\u003c/em\u003e(45), \u003cem\u003evlf1\u003c/em\u003e(59), \u003cem\u003elef4\u003c/em\u003e(67), \u003cem\u003elef5\u003c/em\u003e(75), \u003cem\u003epk-1\u003c/em\u003e(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStructure (34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePolyhedrin\u003c/em\u003e(1), \u003cem\u003ep78/83\u003c/em\u003e(2), \u003cem\u003ep10\u003c/em\u003e(10), \u003cem\u003ep49/49k\u003c/em\u003e(18), \u003cem\u003eodv-e18\u003c/em\u003e(19), \u003cem\u003eodv-ec27\u003c/em\u003e(20), \u003cem\u003eodv-e66\u003c/em\u003e(32), \u003cem\u003eac53\u003c/em\u003e(37), \u003cem\u003evp1054\u003c/em\u003e(40), \u003cem\u003efp25k\u003c/em\u003e(44), \u003cem\u003edesmoplakin\u003c/em\u003e(55), \u003cem\u003eac78\u003c/em\u003e(60), \u003cem\u003egp41\u003c/em\u003e(61), \u003cem\u003eac81\u003c/em\u003e(62), \u003cem\u003etlp20\u003c/em\u003e(63), vp91(64), \u003cem\u003ecg30\u003c/em\u003e(65), \u003cem\u003evp39\u003c/em\u003e(66), \u003cem\u003ep33\u003c/em\u003e(69), \u003cem\u003ep18\u003c/em\u003e(70), \u003cem\u003eodv-e25\u003c/em\u003e(71), \u003cem\u003e38k\u003c/em\u003e(74), \u003cem\u003ep6.9\u003c/em\u003e(76), \u003cem\u003ep40\u003c/em\u003e(77), \u003cem\u003ep12\u003c/em\u003e(78), \u003cem\u003ep45/p48\u003c/em\u003e(79), \u003cem\u003ep87/vp87\u003c/em\u003e(80), \u003cem\u003eodv-ec43\u003c/em\u003e(82), \u003cem\u003ecalyx/pep\u003c/em\u003e(89), \u003cem\u003eparg\u003c/em\u003e(96),\u003cem\u003egp16\u003c/em\u003e(101), \u003cem\u003ep24\u003c/em\u003e(102), \u003cem\u003epkip\u003c/em\u003e(105), \u003cem\u003eF-protein\u003c/em\u003e(115)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOral infectivity (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003epif5/odv-e56\u003c/em\u003e(9), \u003cem\u003ep74/pif0\u003c/em\u003e(15), \u003cem\u003epif6\u003c/em\u003e(53), \u003cem\u003epif4\u003c/em\u003e(73), \u003cem\u003epif3\u003c/em\u003e(94), \u003cem\u003epif2\u003c/em\u003e(106), \u003cem\u003epif1\u003c/em\u003e(114)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAuxiliary (21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ehoar\u003c/em\u003e(4), \u003cem\u003ep26\u003c/em\u003e(11), \u003cem\u003eubiquitin\u003c/em\u003e(25), \u003cem\u003eADPRase\u003c/em\u003e(28), \u003cem\u003edjbp\u003c/em\u003e(35), \u003cem\u003echaB\u003c/em\u003e(43), \u003cem\u003egp37\u003c/em\u003e(46), \u003cem\u003ebro-b\u003c/em\u003e(47), \u003cem\u003echitinase\u003c/em\u003e(48), \u003cem\u003ecathepsin\u003c/em\u003e(50), \u003cem\u003eiap-2\u003c/em\u003e(51), \u003cem\u003emtase\u003c/em\u003e(52), \u003cem\u003ebro-c\u003c/em\u003e(68), \u003cem\u003ep13\u003c/em\u003e(86), \u003cem\u003eiap-1\u003c/em\u003e(88), \u003cem\u003esod\u003c/em\u003e(92), \u003cem\u003eNrk1\u003c/em\u003e(99), \u003cem\u003eptp\u003c/em\u003e(100), \u003cem\u003efgf\u003c/em\u003e(112), \u003cem\u003eegt\u003c/em\u003e(120), \u003cem\u003e38.7k\u003c/em\u003e(123)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAdho30\u003c/em\u003e(12), \u003cem\u003eac145\u003c/em\u003e(21), \u003cem\u003eep23/ac146\u003c/em\u003e(22), \u003cem\u003eac34\u003c/em\u003e(24), \u003cem\u003eAdho3\u003c/em\u003e(30), \u003cem\u003eac43\u003c/em\u003e(33), \u003cem\u003eac52\u003c/em\u003e(36), \u003cem\u003eAdho45\u003c/em\u003e(38), \u003cem\u003eAdho44\u003c/em\u003e(39), \u003cem\u003eASB110/ac55\u003c/em\u003e(41), \u003cem\u003eac59\u003c/em\u003e(42), \u003cem\u003eac75\u003c/em\u003e(57), \u003cem\u003eac76\u003c/em\u003e(58), \u003cem\u003eac108\u003c/em\u003e(83), \u003cem\u003eac110\u003c/em\u003e(81), \u003cem\u003eac117\u003c/em\u003e(90), \u003cem\u003eAdho107\u003c/em\u003e(93), \u003cem\u003eAdho101\u003c/em\u003e(103), \u003cem\u003eac18\u003c/em\u003e(107), \u003cem\u003eac19\u003c/em\u003e(108), \u003cem\u003eAdho113\u003c/em\u003e(113), \u003cem\u003eAdho119\u003c/em\u003e(116), \u003cem\u003eAdho120\u003c/em\u003e(117), \u003cem\u003eAdho121\u003c/em\u003e(118), \u003cem\u003eAdho123\u003c/em\u003e(121), \u003cem\u003eac106\u003c/em\u003e(98), \u003cem\u003enrdB/rr2\u003c/em\u003e(109)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnique (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eorf5\u003c/em\u003e, \u003cem\u003eorf6\u003c/em\u003e, \u003cem\u003eorf7\u003c/em\u003e, \u003cem\u003eorf8\u003c/em\u003e, \u003cem\u003eorf49\u003c/em\u003e, \u003cem\u003eorf87\u003c/em\u003e, \u003cem\u003eorf91\u003c/em\u003e, \u003cem\u003eorf95\u003c/em\u003e, \u003cem\u003eorf97\u003c/em\u003e, \u003cem\u003eorf111\u003c/em\u003e, \u003cem\u003eorf119\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic analysis using 38 baculovirus core genes placed CypoNPV within Alphabaculovirus Group II, indicating a close relationship with CrpeNPV, \u003cem\u003eAdoxophyes honmai\u003c/em\u003e nucleopolyhedrovirus, AdhoNPV, and \u003cem\u003eAdoxophyes orana\u003c/em\u003e nucleopolyhedrovirus, AdorNPV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Comparative genomic analysis further revealed limited collinearity between CypoNPV and CpGV, with the strongest collinearity observed between CypoNPV and CrpeNPV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This finding was supported by synteny analysis, which demonstrated a high degree of conservation in genome orientation and gene order between CypoNPV and CrpeNPV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of viral infections on population parameters of\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate if viral infection severity impacts host performance, \u003cem\u003eC. pomonella\u003c/em\u003e larvae were reared on a standard diet or a formaldehyde-supplemented diet. Then viral load and key life-table parameters between these groups were compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The lower viral group (LV) exhibited a 43.40% reduction in viral copy number compared to the normal viral group (NV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLife-table analysis indicated that this reduction did not enhance population performance; instead, it altered several crucial developmental and reproductive traits. Specifically, the egg period (EP) was significantly shortened by 2.60%, decreasing from 4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 d in NV to 4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 d in LV. In contrast, the pupal duration (PD) was significantly prolonged by 9.31%, from 9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 d to 9.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 d (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Fecundity (F) was significantly reduced by 30.44%, dropping from 107.73\u0026thinsp;\u0026plusmn;\u0026thinsp;7.54 to 74.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36 eggs per female (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e of CypoNPV against first-instar larvae of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(OBs/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% Confidence Interval(OBs/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProbit regression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCorrelation coefficient\u003c/p\u003e \u003cp\u003e(r)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e-1.1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;0.361x-1.627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c2\"\u003e \u003cp\u003e5.2\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c3\"\u003e \u003cp\u003e3.1\u0026times;10\u003csup\u003e4\u003c/sup\u003e-1.8\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;0.165x-0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConsistent with these alterations, no significant differences were observed in the intrinsic rate of increase (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), net reproductive rate (\u003cem\u003eR₀\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), or finite rate of increase (\u003cem\u003eλ\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) between the NV and LV groups. However, the mean generation time (\u003cem\u003eT\u003c/em\u003e) was significantly extended by 6.28%, increasing from 41.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 d to 44.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results indicate that a decreased viral copy number altered host development and reproductive output without conferring a significant demographic advantage to the colony.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLaboratory biological activity of CypoNPV against\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e \u003cb\u003elarvae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo ascertain the insecticidal activity of CypoNPV against \u003cem\u003eC. pomonella\u003c/em\u003e larvae, we evaluated the larval survival in laboratory conditions subsequent to exposure to a gradient of OB concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). CypoNPV exhibited pronounced pathogenicity against first-instar larvae under laboratory conditions. Larval mortality was first observed in the virus-treated groups from 2 days post inoculation (dpi) onwards, while the control group maintained consistent survival throughout the experimental period. A distinct dose-dependent relationship was observed, wherein elevated OB concentrations precipitated an accelerated and more pronounced reduction in larval survival. Specifically, at a concentration of 1.79 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e OBs/mL, survival decreased rapidly, reaching zero by 7 dpi. Conversely, lower concentrations induced a more gradual yet persistent decline in survival over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe estimated median lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) was 3.1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e OBs/mL at 7 dpi and 5.2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e OBs/mL at 14 dpi (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, the median lethal time (LT\u003csub\u003e50\u003c/sub\u003e) decreased proportionally with increasing virus concentration, with the lowest LT\u003csub\u003e50\u003c/sub\u003e recorded at 3.18 days at 1.79 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e OBs/mL (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLT\u003csub\u003e50\u003c/sub\u003e of CypoNPV against first-instar larvae of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(OBs/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLT\u003csub\u003e50\u003c/sub\u003e(d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% Confidence Interval\u003c/p\u003e \u003cp\u003e(d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorrelation Coefficient(r)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.79\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.47\u0026ndash;3.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.79\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.79\u0026ndash;6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.79\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.25\u0026ndash;11.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.79\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.91\u0026ndash;12.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eField control efficacy of CypoNPV against \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFruit damage rate before spraying (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFruit damage rate at 7 days after spraying (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIncrease in fruit damage rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eControl efficacy\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLambda-cyhalothrin\u003c/p\u003e \u003cp\u003esuspension concentrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCypoNPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent lowercase letters within the same column indicate significant differences at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eField control efficacy of CypoNPV against\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e \u003cb\u003epopulations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eField trials indicated that CypoNPV achieved 77.78% control efficacy at 7 days, a level not significantly different from the 86.11% efficacy observed with the chemical insecticide lambda-cyhalothrin (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results suggest that CypoNPV holds significant potential as a biological control agent against \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of dietary formaldehyde supplementation on life-history parameters of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgg duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarval duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePupal rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.67\u0026thinsp;\u0026plusmn;\u0026thinsp;16.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e27.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePupal duration (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdult longevity (days)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0,32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFecundity (eggs/female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e107.73\u0026thinsp;\u0026plusmn;\u0026thinsp;7.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e74.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal preoviposition period (TPOP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e37.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.504\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntrinsic rate of increase (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0059\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNet reproductive rate (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e, eggs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinite rate of increase (\u003cem\u003eλ\u003c/em\u003e, day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean generation time (\u003cem\u003eT\u003c/em\u003e, day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e41.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e44.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: Asterisk (*) indicates a significant difference between the LV and NV groups for the corresponding parameter (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCypoNPV exhibits excellent thermal stability\u003c/h3\u003e\n\u003cp\u003eThermal pre-treatment of CypoNPV did not significantly diminish its biological activity, as evidenced by the substantial retention of infectivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Larvae in the water-treated control group exhibited a normal phenotype. In contrast, larvae infected with untreated CypoNPV at 1.79 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e OBs/mL exhibited a severe phenotype at both 7 and 10 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePre-treatment at 40\u0026deg;C had minimal impact on viral pathogenicity; severe symptoms persisted, with affected larvae ranging from 83.00% to 93.00% at 7 dpi and 83.00% to 97.00% at 10 dpi, and no normal larvae were observed. Conversely, pathogenicity decreased progressively with increasing pre-treatment temperatures. At 60\u0026deg;C, the incidence of severe symptoms reduced to 7.00%\u0026ndash;40.00%, while at 80\u0026deg;C, no severe symptoms were evident, and the majority of larvae remained normal (50.00%-97.00%). Even after pre-treatment at 100\u0026deg;C, residual infectivity was still detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eConsistent with these phenotypic observations, qPCR analysis showed that viral replication was largely maintained following pre-treatment at 40\u0026deg;C, with viral copy numbers remaining at approximately 85.75%-85.79% of the untreated control at 7 dpi. However, viral replication declined at higher temperatures: 65.59%-73.44% at 60\u0026deg;C, 56.17%-65.35% at 80\u0026deg;C, and 43.08%-48.19% at 100\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eLarval mortality showed a similar trend. Pre-treatment at 40\u0026deg;C resulted in 100.00% mortality retention, whereas mortality retention significantly decreased after pre-treatment at 60\u0026deg;C, 80\u0026deg;C, and 100\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThese results demonstrate that CypoNPV possesses robust thermal stability at 40\u0026deg;C and retains detectable pathogenicity even after exposure to higher temperatures.\u003c/p\u003e\n\u003ch3\u003eCypoNPV exhibits considerable UV tolerance\u003c/h3\u003e\n\u003cp\u003eCypoNPV maintained detectable biological activity post-UV irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). As in the thermal stability assay, all larvae treated with water remained phenotypically normal, whereas larvae infected with CypoNPV at 1.79 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e OBs/mL exhibited severe phenotypes at both 7 and 10 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing irradiation at 98 \u0026micro;W/cm\u0026sup2;, a mixed distribution of symptom classes persevered, with severe symptoms affecting 0.00%-16.70% of larvae at 7 dpi and 0.00%-20.00% at 10 dpi, indicating retention of viral pathogenicity. In contrast, symptom severity declined with increasing irradiation intensity. At 180 and 267 \u0026micro;W/cm\u0026sup2;, severe symptoms were infrequent or absent, and the proportion of normal larvae increased to 56.70%-86.60% and 76.70%-90.00% at 7 dpi, and 56.70%-80.00% and 50.00%-86.70% at 10 dpi, respectively. At 384 \u0026micro;W/cm\u0026sup2;, no severe symptoms were observed, with the majority of larvae remained normal (93.30%-96.70% at 7 dpi and 63.30%-90.00% at 10 dpi), although mild or moderate symptoms were still detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eConsistent with these phenotypic observations, qPCR analysis showed that viral replication remained detectable after UV irradiation, albeit at reduced levels with increasing irradiation intensity. At 7 dpi, viral copy numbers were 61.22%-69.76% of the untreated control following irradiation at 98 \u0026micro;W/cm\u0026sup2;, compared to 61.29%-66.29% at 180 \u0026micro;W/cm\u0026sup2;, 61.01%-65.33% at 267 \u0026micro;W/cm\u0026sup2;, and 49.20%-61.75% at 384 \u0026micro;W/cm\u0026sup2; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eLarval mortality showed a similar trend. At 7 dpi, the retention of mortality ranged from 13.33%-53.33% at 98 \u0026micro;W/cm\u0026sup2;, 13.33%-46.67% at 180 \u0026micro;W/cm\u0026sup2;, 10.00%-43.33% at 267 \u0026micro;W/cm\u0026sup2;, and 6.67%-16.67% at 384 \u0026micro;W/cm\u0026sup2;. By 10 dpi, the corresponding ranges were 26.67%-86.67%, 20.00%\u0026ndash;-65.00%, 13.33%-50.00%, and 10.00%-36.67%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThese results indicate that CypoNPV retains significant biological activity post-UV exposure, particularly at lower irradiation intensities, demonstrating considerable UV tolerance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTypically, baculoviruses developed for biological control are more frequently identified from naturally infected field populations or widespread outbreaks rather than from indoor colonies. Although this observation appears atypical, covert baculovirus infections are known to occur regularly in both natural and laboratory insect populations, potentially persisting for extended durations without apparent disease manifestations \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Consequently, the discovery of covertly infected viral strains within a laboratory colony that exhibit pathogenicity after propagation presents a novel avenue for pest biological control. This possibility aligns with growing evidence indicating that agriculturally significant insects harbor diverse and biologically active viromes \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Some of these viruses exhibit host-specific distributions, while others maintain cross-host infectivity, reflecting long-term coevolutionary structuring of viral assemblages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this study, we identified a novel nucleopolyhedrovirus CypoNPV from a stably maintained laboratory population of \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe significant genetic proximity between CypoNPV and CrpeNPV is noteworthy, particularly given that CrpeNPV was initially isolated from a laboratory-reared \u003cem\u003eC. peltastica\u003c/em\u003e population and subsequently demonstrated efficient infectivity toward \u003cem\u003eC. pomonella\u003c/em\u003e. This observation suggests that baculoviruses associated with tortricid hosts can maintain infectivity across related species \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. From an evolutionary perspective, this aligns with the prevailing understanding that while baculoviruses generally exhibit restricted host ranges, host utilization is often conserved among closely related insect species and can be punctuated by host shifts between them \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Consequently, the close relationship between CypoNPV and CrpeNPV could stem from either divergence from a recent common ancestor within a tortricid-associated alphabaculovirus lineage or host-associated speciation following adaptation to distinct, yet phylogenetically related, tortricid hosts \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Based on this evidence, it is reasonable to hypothesize that CypoNPV might also retain pathogenicity against \u003cem\u003eC. peltastica\u003c/em\u003e or other closely related tortricids. However, this remains an inference derived from phylogenetic relatedness and the established cross-infectivity of CrpeNPV, necessitating direct validation through cross-infection bioassays. These findings highlight the importance of considering baculovirus evolution in laboratory colonies not only in terms of persistence but also in relation to long-term host association, tortricid-specific diversification, and potential host shifts among closely related species.\u003c/p\u003e \u003cp\u003eThe isolated CypoNPV, after being multiplied, showed excellent biologically activity and orchard control efficacy against \u003cem\u003eC. pomonella\u003c/em\u003e. While direct LC\u003csub\u003e50\u003c/sub\u003e comparison across baculovirus systems require caution due to variations in host species, larval instar, and bioassay methodology, the rapid kill observed with CypoNPV is comparable to the general efficacy of CpGV, which typically results in susceptible larvae succumbing within 3\u0026ndash;7 dpi \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. CypoNPV does not represent the fastest-acting baculoviruses, as certain \u003cem\u003eS. frugiperda\u003c/em\u003e multiple nucleopolyhedrovirus (SfMNPV) isolates can induce larval mortality under 56 h, but its virulence aligns with that of lepidopteran baculoviruses considered promising candidates for development \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Notably, the orchard efficacy of CypoNPV (77.78%) falls within the established operational range of CpGV products (75\u0026ndash;90%) for \u003cem\u003eC. pomonella\u003c/em\u003e management \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This performance is also consistent with other insect viruses deemed worthy of development, a co-occluded HearNPV mixture caused 89\u0026ndash;100% larval mortality on treated tomato plants and performed comparably to Bacillus thuringiensis and spinosad in reducing fruit damage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Likewise, \u003cem\u003eSpodoptera exigua\u003c/em\u003e multiple nucleopolyhedrovirus (SeMNPV) showed high efficacy in greenhouse sweet pepper crops, where virus-induced mortality of larvae collected from treated plants reached 70\u0026ndash;89%, and recent feeding damage in heavily infested greenhouses declined from 45\u0026ndash;94% before treatment to 0.1\u0026ndash;9.9% after two applications \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Importantly, when evaluated against the lambda-cyhalothrin positive control in the present orchard trial, CypoNPV still provided agriculturally significant population suppression, demonstrating its efficacy not only in laboratory settings but also under orchard conditions. This is particularly relevant given the increasing challenges in \u003cem\u003eC. pomonella\u003c/em\u003e management, including the limited availability of registered insecticides \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, the growing of insecticide resistance cases to lambda-cyhalothrin in field populations \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Furthermore, current commercial virus products for \u003cem\u003eC. pomonella\u003c/em\u003e control are predominantly based on CpGV formulations \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These findings indicate that CypoNPV integrates biologically significant virulence with practical field effectiveness, positioning it as a valuable novel baculoviral resource for codling moth management, especially considering the limited dedicated control options and escalating resistance to conventional insecticides.\u003c/p\u003e \u003cp\u003eThe development and field application of insect-pathogenic viruses depend not only on pathogenicity but also on their thermal sensitivity, and UV sensitivity \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Temperature effects are best understood by considering the microclimate on exposed fruit surfaces where the virus is deposited, rather than solely ambient air temperature. \u003cem\u003eC. pomonella\u003c/em\u003e typically oviposits on fruit surfaces, with newly hatched larvae seeking a suitable location before boring into the fruit. Fruit surface temperatures can significantly exceed air temperature, potentially reaching 15℃ above the daily maximum air temperature under clear summer conditions (Konno et al., 2024). Sunburn browning under sunlight exposure has been linked to fruit surface temperatures between approximately 46\u0026ndash;49℃ \u003csup\u003e40\u003c/sup\u003e. In this study, we found complete retention of CypoNPV activity after a 4-h pre-treatment at 40℃, indicating that CypoNPV can withstand the thermal load that experienced by sun-exposed fruit surfaces throughout the season. Furthermore, the observed decline at 60\u0026ndash;100℃ likely signifies the upper thermal stability limit of the virus, rather than conditions typically encountered in the orchards. Nevertheless, this limit remains relevant for practical applications, as transient heat stress can occur during spray preparation, transport, storage, and on highly exposed plant surfaces post-application. Even after pre-treatment at 100\u0026deg;C, mild symptoms were still observed in a small proportion of larvae, indicating that the virus retained detectable residual infectivity under extreme heat exposure. These findings suggest CypoNPV possesses strong thermal robustness and retains substantial biological activity, further supporting its promise as an effective candidate for practical control.\u003c/p\u003e \u003cp\u003eSurvey shows that monthly mean UV intensities from April to September 2025 in 15 \u003cem\u003eC. pomonella\u003c/em\u003e occurrence regions across China ranged from 339.93 to 398.36 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e (WheatA agricultural meteorological big data platform: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wheata.cn/\u003c/span\u003e\u003cspan address=\"https://wheata.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 1.6.9; accessed on 9 April 2026). In Gaizhou City, Liaoning Province, the field efficacy trial location for this study, the corresponding values ranged from 341.82 to 371.08 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e (Table S2). Consequently, the 384 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e treatments employed in this study align with or approach the upper range of the mean seasonal UV levels documented across \u003cem\u003eC. pomonella\u003c/em\u003e regions, thus representing realistic exposure conditions. Conversely, the lower treatments of 98 and 180 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e can be interpreted as reduced-exposure scenarios, also pertinent to orchard environments, as virus deposits are not constantly subjected to full solar radiation post-application. Against this background, the preservation of measurable infectivity after pretreatment at 98 and 180 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e suggests that CypoNPV can retain activity under lower or partially shielded exposure conditions. In contrast, the observed progressive reduction in activity at 267 and 384 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e indicates that solar radiation likely imposes a more significant limitation on field persistence than temperatures. However, even after UV irradiation, a considerable proportion of larvae still exhibited mild and moderate symptoms, suggesting that CypoNPV retained biologically relevant infectivity and could still cause progressive disease in treated larvae. This pattern is not atypical for baculoviruses. Within \u003cem\u003eC. pomonella\u003c/em\u003e management systems, the limited residual activity of CpGV under orchard conditions has long been recognized as a practical constraint, necessitating frequent reapplication at 7- to 10-d intervals due to rapid solar exposure-induced reduction in persistence on fruit and foliage \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the UV sensitivity of CypoNPV should primarily be addressed as a formulation and application challenge, with future enhancements most likely stemming from the development of solar-protective formulations, canopy-targeted deposition strategies, and optimized spray timing to minimize cumulative UV exposure.\u003c/p\u003e \u003cp\u003eFormaldehyde has historically served as an antiviral or prophylactic agent in insect rearing, particularly against baculoviruses like NPV in lepidopteran colonies \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. To ascertain the impact of CypoNPV infection on population development, we administered formaldehyde through the diet, aiming to eradicate or reduce viral titers. Our findings indicated that while viral copy number decreased by 43.40%, there was no corresponding enhancement in demographic performance; instead, mean generation time increased. This challenges the prevailing assumption that persistent viral load is the primary constraint on host population growth \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similar phenomena have been observed in other insect-virus systems; for instance, ribavirin treatment in Mediterranean fruit flies reduced RNA virus levels without improving host fitness \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and experimental suppression of dengue virus in \u003cem\u003eAedes aegypti\u003c/em\u003e similarly failed to confer fitness benefits \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These results suggest a non-linear relationship between viral burden and host demographic performance. In our study, reducing CypoNPV copy number did not confer a measurable colony-level advantage, reinforcing the conclusion that CypoNPV load was not the principal driver of population development in this laboratory colony. This aligns with broader evidence indicating that latent or persistent insect virus infections do not invariably yield uniform or predictable fitness outcomes \u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReciprocal host-virus selection balances persistence, transmission, and host survival, not necessarily maximal virulence \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Persistent and heritable viruses play a critical role in insect ecology and evolution, moving beyond the notion of incidental infections with minor biological impact \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This broadened perspective is reinforced by recent virome studies, which demonstrate that viral prevalence, abundance, and diversity are modulated by ecological and evolutionary filters, rather than purely stochastic processes \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Moreover, studies on naturally occurring viral infections in laboratory insect populations have shown that persistent viruses can influence host lifespan and reproductive fitness, even in the absence of overt epidemics, suggesting that enduring viral associations retain biological significance under laboratory rearing conditions \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Collectively, the identification of CypoNPV within an indoor population implies that laboratory colonies can harbor latent virus diversity without compromising it biological relevance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These insights advance our comprehension of baculoviruses in insects and provide a more robust framework for integrating viral persistence, coevolution, environmental stability, and the practical deployment of insect viruses developed in laboratory settings. From a practical viewpoint, this study offers novel biological control agents for effective \u003cem\u003eC. pomonella\u003c/em\u003e management and introduces innovative strategies to combat the growing challenge of insecticide resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInsects and Virus\u003c/h2\u003e \u003cp\u003eA laboratory-reared colony of \u003cem\u003eC. pomonella\u003c/em\u003e (LRC) was maintained in a Panasonic incubator ( MLR-352H-PC, Japan) under controlled conditions: 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 16:8 hours (light: dark) photoperiod \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Larvae exhibiting typical symptoms of baculovirus infection were collected and immediately stored at -80\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003cp\u003eTo isolate occlusion bodies (OBs), a crude extract was prepared from symptomatic larvae, followed by OBs purification using 50\u0026ndash;60% sucrose gradient centrifugation, as previously detailed \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElectron Microscopy\u003c/h3\u003e\n\u003cp\u003eFor transmission electron microscope (TEM) examination, purified OBs were placed on carbon-coated grids and negatively stained with 1% aqueous uranyl acetate (w/v), as described previously \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The grids were then examined using a Hitachi HT7700 TEM (Hitachi, Tokyo, Japan) at an 80 kV accelerating voltage.\u003c/p\u003e \u003cp\u003eFor cross-sectional morphological analysis, OBs were embedded in 1% agarose and fixed in 1% osmium tetroxide for 2 h at room temperature. Following fixation, samples were rinsed, dehydrated, embedded, and sectioned. Ultrathin sections (80 nm thick) were prepared using a Leica UC7 ultramicrotome, collected onto 150-mesh copper grids, and double-stained with 2% uranyl acetate and lead citrate \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Finally, the grids were imaged with the Hitachi HT7700 TEM at an accelerating voltage of 100 kV.\u003c/p\u003e\n\u003ch3\u003eGene amplification and complete genome sequencing\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA was extracted from insect samples using the E.Z.N.A.\u0026reg; Viral DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's protocols. Subsequently, partial regions of the \u003cem\u003epolh\u003c/em\u003e/\u003cem\u003egran\u003c/em\u003e, \u003cem\u003elef-8\u003c/em\u003e, and \u003cem\u003elef-9\u003c/em\u003e genes were amplified using degenerate primer pairs (prPH-1/prPH-2, prL8-1/prL8-2, prL9-1/prL9-2), as previously described \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhole-genome sequencing was performed by Sangon Biotech (Shanghai, China) on the Illumina HiSeq platform, generating a total of 47,391,588 raw reads. Quality control and adapter trimming were conducted using Trimmomatic (v0.36) to obtain high-quality clean reads. De novo assembly was performed with SPAdes (v3.5.0). The service provider initially annotated the resulting genome using Prokka (v1.1.0). Open reading frames (ORFs) encoding peptides of \u0026ge;\u0026thinsp;50 amino acids were predicted with ORF Finder. Annotation accuracy was enhanced through a refinement process using closely related nucleopolyhedrovirus genomes, specifically CrpeNPV (accession no. MH394321), as references.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogeny and Kimura 2-parameter analysis\u003c/h2\u003e \u003cp\u003eTo evaluate genetic relationships, protein sequences encoded by CypoNPV were compared with their homologs in reference genomes using BioEdit (v7.0.9.0) with default settings. Multiple genome alignments were conducted using the progressive Mauve algorithm in Geneious v11.0.2 \u003csup\u003e56\u003c/sup\u003e. Gene parity plots were then generated to visualize syntenic relationships and organizational conservation between CypoNPV and other baculoviruses, following established methods \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo infer phylogenetic relationships among baculoviruses, protein sequences of 38 core genes were extracted from 107 complete baculovirus genomes and aligned using MAFFT under the auto strategy and normal alignment mode. Poorly aligned regions were subsequently removed using Gblocks with default parameters. The aligned protein sequences of the 38 core genes were concatenated into a supermatrix using PhyloSuite (version 1.2.2). The optimal partitioning scheme for maximum-likelihood (ML) analysis was determined using PartitionFinder2, based on the Bayesian information criterion (BIC) and employing a greedy search algorithm with linked branch lengths. Finally, an ML tree was constructed with IQ-TREE, utilizing ultrafast bootstrap approximation with 5000 replicates and the SH-aLRT test with 1000 replicates.\u003c/p\u003e \u003cp\u003eFor genetic divergence analysis, pairwise Kimura 2-parameter (K2P) distances were calculated from aligned nucleotide sequences of \u003cem\u003epolh\u003c/em\u003e, \u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e, the concatenated \u003cem\u003epolh\u003c/em\u003e/\u003cem\u003elef-8\u003c/em\u003e/\u003cem\u003elef-9\u003c/em\u003e fragment, and the concatenated 38 core genes using MEGA (version 10.1.8) \u003csup\u003e58\u003c/sup\u003e. Site variation was modeled as uniform, and gaps were treated as pairwise deletions. Species demarcation was conducted using adjusted K2P thresholds: for single or concatenated \u003cem\u003epolh\u003c/em\u003e/\u003cem\u003elef-8\u003c/em\u003e/\u003cem\u003elef-9\u003c/em\u003e genes, thresholds were set as K2P\u0026thinsp;\u0026lt;\u0026thinsp;0.015 (same species), 0.015\u0026ndash;0.050 (uncertain), and \u0026gt;\u0026thinsp;0.050 (different species). In contrast, for the concatenated 38 core-gene fragments, the corresponding thresholds were K2P\u0026thinsp;\u0026lt;\u0026thinsp;0.021 (same species), 0.021\u0026ndash;0.072 (uncertain), and \u0026gt;\u0026thinsp;0.072 (different species), following criteria established previously \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of viral copy numbers\u003c/h2\u003e \u003cp\u003eTotal viral DNA was extracted from the larvae using the EasyPure Viral DNA/RNA Kit. Samples were processed in groups of 3\u0026ndash;4 individuals, with three replicates per condition. The extracted DNA was eluted in 30 \u0026micro;L of sterile ddH₂O. The \u003cem\u003epolyhedrin\u003c/em\u003e gene, a conserved core gene of CypoNPV, was selected as the target for quantifying viral replication due to its consistent correlation with viral genomic copies. A quantitative standard was constructed by amplifying the \u003cem\u003epolyhedrin\u003c/em\u003e gene via PCR, followed by gel extraction, cloning into a pMD\u0026trade;-19 vector, and transformation. Positive clones (designated T-polh) were verified by sequencing, cultured, and subjected to plasmid extraction. The plasmid concentration (ng/\u0026micro;L) was measured for copy number calculation.\u003c/p\u003e \u003cp\u003eViral copy number was calculated using the following formula:\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cimg 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\" width=\"543\" height=\"61\"\u003e\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePopulation parameters of infected with different concentrations of viruses\u003c/h2\u003e \u003cp\u003eFor life-table analysis, newly laid eggs from the laboratory colony were divided into two groups: the normal viral group (NV), fed a standard diet, and the lower viral group (LV), fed a formaldehyde-supplemented diet. In the LV group, formaldehyde was incorporated into the artificial diet at approximately three times the original formulation's level, specifically 200 \u0026micro;L formaldehyde per 50 mL diet. This concentration was determined from preliminary experiments, as it effectively reduced viral copy number without significantly impacting survival rates. Three replicates of 15 eggs each were randomly assigned to each treatment and maintained under identical environmental conditions. Individuals were monitored daily to record the developmental duration of each life stage and their survival status. Upon hatching, larvae were provided with an artificial diet, which was replaced every 5 days until pupation. Pupal duration and adult emergence were recorded, and newly emerged adults were paired and kept under the same rearing conditions for oviposition. Fecundity and adult survival were documented daily until all individuals had expired.\u003c/p\u003e \u003cp\u003eData analysis was carried out Population parameters, including the intrinsic rate of increase (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e), finite rate of increase (\u003cem\u003eλ\u003c/em\u003e), net reproductive rate (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e), and mean generation time (\u003cem\u003eT\u003c/em\u003e), were calculated using the TWOSEX-MSChart software and bootstrap methods for standard error estimation \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory biological activity of CypoNPV\u003c/h2\u003e \u003cp\u003eTo evaluate the biological activity of purified CypoNPV OBs against neonate larvae, we employed a droplet-feeding method (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). OB concentrations were determined using a hemocytometer (Thoma cell counting chamber), and a six-point, ten-fold serial dilution series was prepared, resulting in doses ranging from 1.79 \u0026times; 10⁶ to 1.79 \u0026times; 10\u0026sup1; OBs/mL. For each concentration, 2 \u0026micro;L of OB suspension was applied to the surface of an artificial diet cube (0.5 \u0026times; 0.5 \u0026times; 2 cm). After air-drying, the diet blocks were placed into individual wells of a 24-well plate. Newly hatched larvae (\u0026lt;\u0026thinsp;8 h old) were introduced to the treated diet and kept under standard rearing conditions. After 24 h, larvae were transferred to fresh, untreated diet, and any deceased individuals were removed. Each dose was tested on 30 neonate larvae, with sterile water serving as the negative control. The entire bioassay was repeated three times independently. Plates were incubated at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and larval mortality was recorded daily for 15 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eField control efficacy of CypoNPV\u003c/h2\u003e \u003cp\u003eTo further evaluate CypoNPV insecticidal efficacy under field conditions, we conducted a trial in an apple orchard located in Wumeifang Village, Jiuzhai Town, Gaizhou City, Liaoning Province, China (40\u0026deg;3\u0026rsquo;5\u0026rsquo;\u0026rsquo; N, 122\u0026deg;5\u0026rsquo;44\u0026rsquo;\u0026rsquo; E) from August 14 to August 22, 2024. Before treatment, we assessed the number of infested fruits to establish a baseline. CypoNPV was applied at a concentration of 1.79 \u0026times; 10⁵ OBs/mL using a motorized sprayer during evening hours, ensuring even coverage on the leaf surface until runoff. Each treatment, including the untreated control, consisted of six apple trees, with a total of 12 L of spray solution applied per treatment. Alongside the viral treatment, we included a negative control (water) and a positive control consisting of 25 g/L lambda-cyhalothrin emulsifiable concentrate (EC) (Syngenta, China), diluted 3000-fold with water and applied using a backpack sprayer. Post application, fruit damage was evaluated by examining over 25 fruits per tree from each of the four cardinal directions (east, south, west, and north). Field control efficacy was calculated at 7 days post-application, according to the Chinese national standard GB/T 17980.65\u0026ndash;2004.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThermal and ultraviolet pretreatment of CypoNPV\u003c/h2\u003e \u003cp\u003ePurified CypoNPV OB suspensions were adjusted to 1.79 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e OBs/mL and used for thermal and ultraviolet (UV) stability assays. For the thermal treatment, aliquots of the viral suspension were incubated in a metal bath at 40, 60, 80, or 100\u0026deg;C for 1, 2, 3, or 4 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). For the UV treatment, viral suspensions at the same concentration were exposed to UV intensities of 98, 180, 267, or 384 \u0026micro;W/cm\u0026sup2; for 1, 2, 3, or 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To minimize the shielding effect of the water layer, 0.3 mL of viral suspension was first dispensed into a Petri dish and air-dried at room temperature in the dark before irradiation. The untreated viral suspension served as the control group. Subsequently, the treated virus was then subjected to the same third-instar feeding bioassay described above, with 20 larvae per treatment and three independent replicates, as third-instar larvae provided enhanced visibility and differentiation of symptom progression Infection symptoms and larval mortality were recorded at 7 and 10 days post-inoculation. For each time point, 9\u0026ndash;12 larvae were collected, weighed, and stored at -20\u0026deg;C for subsequent viral DNA extraction. This weighing enabled later normalization of viral copy number per unit body weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe log-probit regression equation and LC\u003csub\u003e50\u003c/sub\u003e (and LT\u003csub\u003e50\u003c/sub\u003e) value were determined by probit analysis using SPSS 26.0. The results were graphically represented using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA), with all values being expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Furthermore, significant difference in field efficacy between CypoNPV and control was analyzed through independent samples \u003cem\u003et\u003c/em\u003e-tests using SPSS 26.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Liaoning Distinguished Youth Scholars Science Foundation (2024JH3/50100027), National Key Research and Development Program (2021YFD1400201), and the Education Department of Liaoning Province (JYTYB2024038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.X.L.: investigation, data curation, and writing-original draft; H.J.Z.: methodology, sample processing and data acquisition; B.K.W.: sample processing and data acquisition; Y.F.: sample processing and data acquisition; B.B.: sample processing; P.G.: investigation; Y.T.L.: investigation, supervision, conceptualization, and funding acquisition; X.Q.Y.: supervision, conceptualization, funding acquisition, and writing-review and editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDaugherty, M. 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Total Environ.\u003c/em\u003e 913, 169643 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9410995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9410995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile naturally occurring insect-pathogenic viruses are often highly pathogenic and utilized as biocontrol agents, the potential of laboratory-maintained viruses remains underexplored. We report a novel nucleopolyhedrovirus, CypoNPV, exhibiting excellent biologically activity against its host, \u003cem\u003eCydia pomonella\u003c/em\u003e. CypoNPV is genetically similar to \u003cem\u003eCryptophlebia peltastica\u003c/em\u003e nucleopolyhedrovirus (CrpeNPV), which also infects \u003cem\u003eC. pomonella\u003c/em\u003e. Reducing viral copy numbers with formaldehyde did not enhance host population development. However, proliferated CypoNPV maintained high pathogenicity against newly hatched \u003cem\u003eC. pomonella\u003c/em\u003e larvae. Field trials confirmed its excellent efficacy, comparable to the chemical insecticide lambda-cyhalothrin. Furthermore, CypoNPV displayed robust thermal and UV resistance; virulence was unaffected by 40\u0026deg;C treatment. Exposure to 60\u0026deg;C or UV (267 \u0026micro;W/cm\u0026sup2;) for 1\u0026ndash;4 hours decreased larval mortality to 30.00%-76.67% and 10.00%-43.33%, respectively. These findings enhance our understanding of baculovirus persistence in insects and their coevolution, thereby establishing a new avenue for the discovery and application of pathogenic viruses for pest biological control.\u003c/p\u003e","manuscriptTitle":"A new nucleopolyhedrovirus isolated from a laboratory population of Cydia pomonella exhibits excellent biological activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 06:27:15","doi":"10.21203/rs.3.rs-9410995/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10d72bc5-6d01-46be-a3fa-cd08292c54eb","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[{"type":"editorAssigned","content":"","date":"2026-05-08T04:15:04+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66479162,"name":"Biological sciences/Microbiology"},{"id":66479163,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-22T06:27:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 06:27:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9410995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9410995","identity":"rs-9410995","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}