Entomopathogenic viruses, such as baculoviruses and cypoviruses, have been employed as biological pesticides against agricultural and forestry pests. However, their susceptibility to inactivation under field UV radiation has hindered their broader application. In this study, we effectively improved the UV resistance of insect virus occlusion bodies (OBs) by coating their surfaces with silica nanoparticles (SiO2 NPs). Monodisperse SiO2 NPs with uniform size distribution and excellent colloidal stability were synthesized using the Stöber method. Subsequent amination modification of the SiO2 NPs with a silane coupling agent shifted their isoelectric point from 3.2 to 8.1. This modification imparted a strong positive charge to the NPs within the pH range of 4.5-5.5, while the OBs of insect viruses remained negatively charged in this range. Consequently, the amino-functionalized SiO2 NPs were successfully coated onto the surfaces of OBs of three representative insect viruses: nucleopolyhedrovirus, granulovirus, and cypovirus, through electrostatic interactions. Laboratory bioassays confirmed that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) coated with SiO2-NH2 NPs retained its native viral pathogenicity against Spodoptera exigua larvae under normal laboratory condition, while it demonstrated 2.299-2.712 folds higher potency than MbMNPV physically mixed with unmodified SiO2 NPs after UV irradiation. Outdoor trials revealed that SiO2-NH2 NPs coating significantly improved the survival time of MbMNPV, with the median survival time increased from 1.43 days to 5.15 days. This nanoparticle coating strategy establishes a robust platform for developing photostable biopesticides while preserving their ecological safety profiles. The modular nature of this approach suggests its broad applicability across different entomopathogenic virus formulations.

Bacillus anthracis is the causative agent of anthrax disease. Our prior research indicated that cyclic (c)-di-AMP accumulation attenuated bacterial virulence in mice. However, the function of c-di-AMP secretion and its regulatory mechanism in anthrax have not been revealed. We characterized the role of the ecto-nucleotidase CpdB in B. anthracis. CpdB exhibits phosphodiesterase activity towards c-di-AMP and 2ʹ3ʹ-cGMP-AMP, and nucleotidase activity towards several mononucleotides, including the preferred substrates c-di-AMP and pApA. Our results demonstrated that inactivation of cpdB altered purine nucleotide metabolism, decreasing the levels of anthrax toxins and extracellular proteases while increasing the expression of adhesion factor BslA. The rates of adhesion and invasion of B. anthracis to both endothelial cells and immune cells in vitro were enhanced by cpdB inactivation. In infected silkworms, cpdB inactivation led to higher levels of colonization in hemolymph, but virulence was attenuated. These findings suggest that cpdB has pleiotropic functions in the infection and virulence of B. anthracis.

BACKGROUNDBaculoviruses are ideal biological insecticides, providing long-lasting pest control and environmental benefits. Alphabaculovirus mabrassicae stains, with their broad host range, have been effective in agricultural pest management. Various A. mabrassicae isolates (MbMNPV-CHb1/CTa/K1/QD, MyseMNPV-Hb, HearMNPV and MacoNPV-B) have been identified in different hosts. Identifying more effective A. mabrassicae strains with detailed genetic information is crucial for commercial use.RESULTSLaboratory bioassays showed that the median lethal concentration (LC50) of MyseMNPV-Hb against Mythimna separata was significantly lower than those against Helicoverpa armigera and Spodoptera exigua, but higher than the LC50 of MbMNPV-CHb1, MbMNPV-QD and HearMNPV against H. armigera or S. exigua. Comparative genomic analysis revealed significant differences in genomic composition and single-nucleotide polymorphisms between MyseMNPV-Hb and the other isolates. A piggyBac-like element, likely to have been from Alcis repandata (Lepidoptera: Geometridae), was identified in the genomes of these isolates. Eight genes in the A. mabrassicae genomes were found to be under positive selection.CONCLUSIONAlphabaculovirus mabrassicae isolates exhibit different infectivity in various pests, indicating the need for selecting appropriate isolates specific target pests. This study elucidates the genetic factors contributing to the differential infectivity of A. mabrassicae isolates and extends knowledge on its population characteristics. (c) 2025 Society of Chemical Industry.

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV- QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV- Hb), respectively. The Kimura two- parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV- Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV- B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein- coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV- Hb, HearMNPV, and MacoNPV- B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.

ABSTRACT The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis . By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis . Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis. IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis . c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

MicroRNAs (miRNAs) represent a class of short, non-coding RNAs that are widely acknowledged as crucial participants in virus-host interactions. MiR-184, a highly conserved and abundant miRNA in insects, has yet to be extensively studied for its involvement in baculovirus infection. In this study, we investigated how miR-184 affects the infection and replication of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The results indicated that after AcMNPV infection, there was an initial increase in the expression of miR-184 within 24 h, followed by a subsequent decrease. MiR-184 can inhibit AcMNPV's DNA replication and budded virus production by directly targeting four viral genes, namely ie1, ac66, p49, and lef9. Moreover, suppressing miR-184 expression enhanced the insecticidal efficacy of AcMNPV against Spodoptera exigua larvae and markedly elevated the host ATPase gene expressions. These findings showed that miR-184 had a substantial impact on the interactions between baculoviruses and insects, presenting a prospective candidate for developing highly effective miRNA-based biopesticides.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is sur-rounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during forma-tion of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos- tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi -point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.

BACKGROUND:Baculoviruses act as effective biological control agents against the invasive pest Hyphantria cunea Drury. In this study, two Chinese Hyphantria cunea nucleopolyhedrovirus (HycuNPV) isolates, HycuNPV-BJ and HycuNPV-HB, were deep sequenced and compared with the Japanese isolate, HycuNPV-N9, to determine whole-genome level diversity and evolutionary history.RESULTS:The divergence of the phylogenetic tree and the K2P distances based on 38 core-gene concatenated alignment revealed that two Chinese HycuNPV isolates were a novel species of Alphabaculovirus that infected Hyphantria cunea in China. The gene contents indicated significant differences in the HycuNPV genomes between the Chinese and Japanese isolates. The differences included gene deletions, acquisitions and structural transversions, but the main difference was the high number of single nucleotide polymorphisms (SNPs). In total, 10,393 SNPs, corresponding to approximately 8% of the entire HycuNPV-N9 genome sequence, were detected in the aligned reads. By analyzing non-synonymous variants, we found that hotspot mutation-containing genes had mainly unknown functions and most were early expressing genes. We found that the hycu78 gene which had early and late promoter was under positive selection. Biological activity assays revealed that the infectivity of HycuNPV-HB was greater than that of HycuNPV-BJ, and the killing speed of HycuNPV-HB was faster than that of HycuNPV-BJ. A comparison of molecular genetic characteristics indicated that the virulence differences between the two isolates were affected by SNP and structural variants, especially the homologous repeat regions.CONCLUSIONS:The genomes of the two Chinese HycuNPV isolates were characterized, they belonged to a novel species of Alphabaculovirus that infected Hyphantria cunea in China. We inferred that the loss or gain of genetic material in the HycuNPV-HB and HycuNPV-BJ genomes resulted in new important adaptive capabilities to the H. cunea host. These results extend the current understanding of the genetic diversity of HycuNPV and will be useful for improving the applicability of this virus as a biological control agent.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.

2020年10月ICTV更新了主要病毒名录,该文件新增了纳尔达病毒纲Naldaviricetes和勒乏病毒目Lefavirales。勒乏病毒目含中有包括杆状病毒科Baculoviridae在内的3个科,而杆状病毒科分为4个属:甲型杆状病毒属Alphabaculovirus、乙型杆状病毒属Betabaculovirus、丙型杆状病毒属Deltabaculovirus和丁型杆状病毒属Gammabaculovirus,该名录中杆状病毒科有85个主要种。根据ICTV网站病毒名称和种名的书写规范,杆状病毒的种名由其宿主昆虫的种名和病毒特性词（如nucleopolyhedrovirus和granulovirus）组成,其中宿主昆虫的属名首字母大写,其余字母均小写。病毒的种名（在用于系统分类时）书写全部用斜体,但其他情形时病毒名称（包括其宿主的属和种名）均采用正体书写。杆状病毒名称的缩写一般采用宿主属名和种名的前两位双字母+病毒特性词缩写的方式,但16种传统杆状病毒的缩写仍沿用宿主的属名和种名首位单字母+病毒特性词的缩写的方式。

AbstractThe median lethal dose (LD50) is commonly used to indicate acute toxicity of an insecticide to an insect species. Approximate confidence intervals for LD50s are often calculated using the Fieller and delta methods. It is often necessary to compare the relative potencies of several insecticides with a population or of one insecticide with different populations. Comparing the LD50s using probit/logit–log(dose) regressions with parallel slopes can be implemented in many software packages, but for the cases with arbitrary slopes are not generally available. We used the glm function in R to calculate and compare lethal doses without assuming equal slopes. Bioassay datasets from the literature fitted using the logit model gave the 95% confidence limits (95% CLs) for the lethal doses using Fieller’s theorem and incorporating a heterogeneity factor identical to the 95% CLs determined using the PoloPlus software. The delta method gave 95% CLs identical to the 95% CLs determined using the R drc package. The same datasets fitted using the probit model gave 95% CLs similar to the 95% CLs determined using PoloPlus and the drc package. The natural response rates for the control group were included using Abbott’s equation. When the potency ratio method and the z-test were used to identify differences between two lethal doses, and when the χ2 and log likelihood ratio tests were used to determine whether the regression lines were parallel, the conclusions were the same as those gave by PoloPlus and the drc package.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene that is essential for AcMNPV propagation. However, the key domains or residues of the AC75 protein that play a role in viral propagation have not been identified. In this study, sequence alignment revealed that residues Phe-54 and Gln-81 of AC75 were highly conserved among alphabaculoviruses and betabaculoviurses. Thus, Phe-54 and Gln-81 AC75 mutation bacmids were constructed. We found that Gln-81 was not required for viral propagation, whereas mutating Phe-54 reduced budded virus production by 10-fold and impaired occlusion body formation when compared with that of the wild-type AcMNPV. Electron microscopy observations showed that the Phe-54 mutation affected polyhedrin assembly and also occlusion-derived virus embedding, whereas western blot analysis revealed that mutating Phe-54 reduced the amount of AC75 but did not affect the localization of AC75 in infected cells. A protein stability assay showed that the Phe-54 mutation affected AC75 stability. Taken together, Phe-54 was identified as an important residue of AC75, and ac75 is a pivotal gene in budding virus production and occlusion body formation.

The novel severe acute respiratory syndrome coronavirus 2 （SARS-Co V-2） has caused an outbreak of COVID-19 （2019coronavirus infectious disease） that has led to a global public health crisis.The spread of SARS-Co V-2 can be rapid,but there is still considerable controversy about its main route of transmission[1,2];that is,

The fall armyworm, Spodoptera frugiperda, is a new invading pest in China. The baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a pathogenic agent of the fall armyworm and a potential agent for its control in integrated pest management strategies. In this work, we analyze the molecular and biological characteristics of an SfMNPV isolate collected from maize in China (SfMNPV-Hub). Two genotypes were further isolated from SfMNPV-Hub by an in vivo cloning method. The PstI profile of one genotype (SfHub-A) was similar to genotype A of the SfMNPV Colombian isolate, and the other (SfHub-E) was similar to genotype E of the Colombian isolate. The bioactivity of SfHub-A against second-instar S. frugiperda larvae was not significantly different from that of SfMNPV-Hub, whereas SfHub-E was 2.7–5.5 fold less potent than SfMNPV-Hub. The speed of kill of SfHub-E was quicker than SfMNPV-Hub, while SfHub-A acted slower than SfMNPV-Hub. Occlusion body (OB) production of SfHub-A in an S. frugiperda cadaver was significantly higher than that of SfMNPV-Hub, while SfHub-E yielded far fewer occlusion bodies (OBs) in the host larvae. These results provide basic information for developing a virus-based pesticide against the invading pest S. frugiperda.

Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8-fold higher c-di-AMP levels than in the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, excess c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpH in trans from a Pspac promoter restored bacterial growth, virulence factor expression, and resistance to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction. IMPORTANCE Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. Vegetative cells of this species produce anthrax toxin proteins and S-layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that excess elevated c-di-AMP levels inhibit bacterial growth and reduce expression of S-layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.

Dear Editor The most important property of a virus is its infectivity.To measure infectivity,one can assay viral replication in cells to obtain a titer for a given virus stock.A titer is defined as a given number of infectious viral units per unit volume,and an infectious unit is the smallest amount of virus that produces recognizable effects[e.g.,cytopathic effect(CPE),dot blot immunoreactivity].The median tissue culture infectious dose(TCID50)is defined as the dilution of a virus required to infect 50％ of a given cell culture.Several methods have been developed to calculate the TCID50 including the Spearman-K?rber method(Spear-man 1908;K?rber 1931),the Reed-Muench method(Reed and Muench 1938),the improved K?rber method(Sun 1963),the Weil method(Meynell GG and Meynell E 1970),and probit/logit regression models(Finney 1971).In this letter,we analyze datasets using three popular methods for calculating the TCID50 in Excel based calculator(Supplementary file 1).We also compared these results with those of probit/logit regression models and discuss the differences among these methods.

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Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5 and 3 untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

The Cydia pomonella granulovirus (CpGV) GP37 has synergistic effects on the infectivity of nucleopolyhedroviruses (NPVs), however, the mechanism employed is unclear. In this study, in vitro and in vivo binding assays indicated that GP37 efficiently bound to the midgut peritrophic membrane (PM) of Spodoptera exigua larvae. Treatment with GP37 led to the damage of the PM's compacted structure and the generation of the PM perforations, and the enhancement of the PM's permeability. qPCR results further demonstrated that GP37 increased the ability of occlusion-derived virions (ODV) to cross the PM. R18-labeling experiments exhibited that GP37 also promoted the fusion of ODVs and insect midgut epithelia. Altogether, our present results revealed that the synergistic mechanism of GP37 to the infectivity of NPV might involve two parts. GP37 damaged the integrity of the PM after binding, which enhanced the PM's permeability and increased the ability of ODVs to cross the PM, finally facilitating the ODVs reaching the midgut. In addition, GP37 promoted the fusion of ODVs and insect midgut epithelia. Our data expand the understanding of the mechanism used by baculovirus synergistic factors and provide a foundation for the development of high-efficiency baculoviral insecticides.