Hepatocellular carcinoma (HCC) is a highly vascularized carcinoma, and targeting its neovascularization represents an effective therapeutic approach. Our previous study demonstrated that the baculovirus-mediated endostatin and angiostatin fusion protein (BDS-hEA) effectively inhibits the angiogenesis of vascular endothelial cells and the growth of HCC tumors. However, the mechanism underlying its anti-angiogenic effect remains unclear. Increasing evidence suggests that autophagy has a significant impact on the function of vascular endothelial cells and response to cancer therapy. Hence, the objective of this research was to investigate the correlation between BDS-hEA-induced angiogenesis inhibition and autophagy, along with potential regulatory mechanisms. Our results demonstrated that BDS-hEA induced autophagy in EA.hy926 cells, as evidenced by the increasing number of autophagosomes and reactive oxygen species, accompanied by an upregulation of Beclin-1, LC3-II/LC3-I, and p62 protein expression. Suppression of autophagy using 3-methyladenine attenuated the functions of BDS-hEA-induced EA.hy926 cells, including the viability, proliferation, invasion, migration, and angiogenesis. Moreover, BDS-hEA induced autophagy by downregulating the expression of CD31, VEGF, and VEGFR2, as well as phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR), while concurrently upregulating phosphorylated AMP-activated protein kinase (p-AMPK). The in vivo results further indicated that inhibition of autophagy by chloroquine significantly impeded the ability of BDS-hEA to suppress HCC tumor growth in mice. Mechanistically, BDS-hEA prominently facilitated autophagic apoptosis in tumor tissues and decreased the levels of ki67, CD31, VEGF, MMP-9, p-AKT, and p-mTOR while simultaneously enhancing the p-AMPK expression. In conclusion, our findings suggest that BDS-hEA induces autophagy as a cytotoxic response by modulating the AMPK/AKT/mTOR signaling pathway, thereby exerting anti-angiogenic effects against HCC.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the Baculoviridae family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by Spodoptera frugiperda (Sf) insect cells were characterised for the first time. Using S. frugiperda (SfC1B5) cells stably expressing the baculovirus gp64, the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking p6.9 (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101-which may be useful as a protein marker for sEVs.

Various isolates of the Cydia pomonella granulovirus (CpGV) are used as insect pest control agents against codling moth (CM, Cydia pomonella L.), a predominant pest in apple orchards. Three different types (I-III) of dominantly inherited field resistance of CM larvae to CpGV have been recently identified. In this study, transcription of virus genes in midgut cells of type II-resistant CM larvae infected with different CpGV isolates, i.e., CpGV-M and CpGV-S (both prone to type II resistance) as well as CpGV-E2 (breaking type II resistance) was determined by strand-specific RNA sequencing (RNA-Seq) at an early infection stage (72 h post infection). Based on principal component analysis of read counts and the quantitative distribution of single nucleotide polymorphisms (SNPs) in the RNA-Seq data, a bioinformatics analysis pipeline was developed for an a posteriori identification of the infective agents. We report that (i) identification of infective agent is crucial, especially in in vivo infection experiments, when activation of covert virus infections is a possibility, (ii) no substantial difference between CpGV-M and CpGV-S transcription was found in type II-resistant CM larvae despite a different resistance mechanism, (iii) the transcription level of CpGV-M and CpGV-S was much lower than that of CpGV-E2, and (iv) orf59 (sod), orf89 (pif-6), orf92 (p18), and orf137 (lef-10) were identified as significantly downregulated genes in resistance-prone isolates CpGV-M and CpGV-S. For type II resistance of CM larvae, we conclude that CpGV-M and CpGV-S are both able to enter midgut cells, but viral transcription is significantly impaired at an early stage of infection compared to the resistance-breaking isolate CpGV-E2.
OBJECTIVE: CpGV is a highly virulent pathogen of codling moth, and it has been developed into one of the most successful commercial baculovirus biocontrol agents for pome fruit production worldwide. The emergence of field resistance in codling moth to commercial CpGV products is a threat toward the sustainable use of CpGV. In recent years, different types of resistance (type I-III) were identified. For type II resistance, very little is known regarding the infection process. By studying the virus gene expression patterns of different CpGV isolates in midguts of type II-resistant codling moth larvae, we found that the type II resistance mechanism is most likely based on intracellular factors rather than a receptor component. By applying SNP mapping of the RNA-Seq data, we further emphasize the importance of identifying the infective agents in in vivo experiments when activation of a covert infection cannot be excluded.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Although it is generally believed that large DNA viruses capture genes by horizontal gene transfer (HGT), the detailed manner of such transfer has not been fully elucidated. Here, we searched for genes in the coleopteran entomopoxvirus (EV) Anomala cuprea entomopoxvirus (ACEV) that might have been gained by ACEV by HGT. We classified the potential source organisms for HGT into three categories: the host A. cuprea; other organisms, including viruses unrelated to EVs; and organisms with uncertain host attribution. Of the open reading frames (ORFs) of the ACEV genome, 2.1 % were suggested to have been gained from the host by ACEV or its recent ancestor via HGT; 8.7 % were possibly from organisms other than the host, and 3.7 % were possibly from the third category of organisms via HGT. The analysis showed that ACEV contains some interesting ORFs obtained by HGT, including a large ATP-binding cassette protein (ABC transporter) ORF and a tenascin ORF (IDs ACV025 and ACV123, respectively). We then performed a detailed analysis of the HGT of the ACEV large ABC transporter ORF-the largest of the ACEV ORFs. mRNA sequences obtained by RNA-seq from fat bodies-sites of ACEV replication-and midgut tissues-sites of initial infection-of the virus\'s host A. cuprea larvae were subjected to BLAST analysis. One type of ABC transporter ORF from the fat bodies and two types from the midgut tissues, one of which was identical to that in the fat bodies, had the greatest identity to the ABC transporter ORF of ACEV. The two types from the host had high levels of identity to each other (approximately 95 % nucleotide sequence identity), strongly suggesting that the host ABC transporter group consisting of the two types was the origin of ACV025. We then determined the sequence (12,381 bp) containing a full-length gene of the A. cuprea ABC transporter. It turned out to be a transcription template for the abovementioned mRNA found in both tissues. In addition, we determined a large part (ca. 6.9 kb) of the template sequence for the mRNA found only in the midgut tissues. The results showed that the ACEV ABC transporter ORF is missing parts corresponding to introns of the host ABC transporter genes, indicating that the ORF was likely acquired by HGT in the form of mRNA. The presence of definite duplicated sequences adjacent to the ACEV ABC transporter genes-a sign of LINE-1 retrotransposon-mediated HGT-was not observed. An approximately 2-month ACV025 transcription experiment suggested that the transporter sequence is presumed to be continuously functional. The amino acid sequence of ACV025 suggests that its product might function in the regulation of phosphatide in the host-cell membranes.

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection.
OBJECTIVE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Cnaphalocrocis medinalis granulovirus (CnmeGV), belonging to Betabaculovirus cnamedinalis, can infect the rice pest, the rice leaf roller. In 1979, a CnmeGV isolate, CnmeGV-EP, was collected from Enping County, China. In 2014, we collected another CnmeGV isolate, CnmeGV-EPDH3, at the same location and obtained the complete virus genome sequence using Illumina and ONT sequencing technologies. By combining these two virus isolates, we updated the genome annotation of CnmeGV and conducted an in-depth analysis of its genome features. CnmeGV genome contains abundant tandem repeat sequences, and the repeating units in the homologous regions (hrs) exhibit overlapping and nested patterns. The genetic variations within EPDH3 population show the high stability of CnmeGV genome, and tandem repeats are the only region of high genetic variation in CnmeGV genome replication. Some defective viral genomes formed by recombination were found within the population. Comparison analysis of the two virus isolates collected from Enping showed that the proteins encoded by the CnmeGV-specific genes were less conserved relative to the baculovirus core genes. At the genomic level, there are a large number of SNPs and InDels between the two virus isolates, especially in and around the bro genes and hrs. Additionally, we discovered that CnmeGV acquired a segment of non-ORF sequence from its host, which does not provide any new proteins but rather serves as redundant genetic material integrated into the viral genome. Furthermore, we observed that the host\'s transposon piggyBac has inserted into some virus genes. Together, dsDNA viruses could acquire non-coding genetic material from their hosts to expand the size of their genomes. These findings provide new insights into the evolution of dsDNA viruses.

The composition of microbiota in the digestive tract gut is essential for insect physiology, homeostasis, and pathogen infection. Little is known about the interactions between microbiota load and oral infection with baculoviruses. CnmeGV is an obligative baculovirus to Cnaphalocrocis medinalis. We investigated the impact of CnmeGV infection on the structure of intestinal microbes of C. medinalis during the initial infection stage. The results revealed that the gut microbiota profiles were dynamically driven by pathogen infection of CnmeGV. The numbers of all the OTU counts were relatively higher at the early and later stages, while the microbial diversity significantly increased early but dropped sharply following the infection. The compositional abundance of domain bacteria Firmicutes developed substantially higher. The significantly enriched and depleted species can be divided into four groups at the species level. Fifteen of these species were ultimately predicted as the biomarkers of CnmeGV infection. CnmeGV infection induces significant enrichment of alterations in functional genes related to metabolism and the immune system, encompassing processes such as carbohydrate, amino acid, cofactor, and vitamin metabolism. Finally, the study may provide an in-depth analysis of the relationship between host microbiota, baculovirus infection, and pest control of C. medinalis.