Baculoviruses are entomopathogens that carry large, double-stranded circular DNA genomes and infect insect larvae of Lepidoptera, Hymenoptera and Diptera, with applications in the biological control of agricultural pests, in the production of recombinant proteins and as viral vectors for various purposes in mammals. These viruses have a variable genetic composition that differs between species, with some sequences shared by all known members, and others that are lineage-specific or unique to isolates. Based on the analysis of nearly 300 sequenced genomes, a thorough bioinformatic investigation was conducted on all the baculoviral protein coding sequences, characterizing their orthology and phylogeny. This analysis confirmed the 38 protein coding sequences currently considered as core genes, while also identifying novel coding sequences as candidates to join this set. Accordingly, homology was found among all the major occlusion body proteins, thus proposing that the polyhedrin, granulin and CUN085 genes be considered as the 39th core gene of Baculoviridae.

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV), has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny.Methods: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in a comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined.Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells.Conclusion: Consequently, this insect pathogen is proposed as an alternative to non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

Background: Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T‐box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv‐Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. Methods and Results: Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv‐Tbx20 or Bv‐Null (control). Subsequently, in sheep with AMI, Bv‐Tbx20 or Bv‐Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv‐Tbx20 showed increased Tbx20 , promitotic and angiogenic gene expression, decreased levels of P21 , increased Ki67‐ (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm ² , P <0.05) and PHH3 (phospho‐histone H3)‐labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm ² , P <0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm ² , P <0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm ² , P <0.05) densities. At 30 days, Bv‐Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P <0.05) and slightly improved left ventricular function. Baculoviral gene transfer‐mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects in vitro. Conclusions: In sheep with AMI, Bv‐Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv‐Tbx20 gene therapy may contribute to cardiac regeneration following AMI.

Background/Objectives: Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10⁴ virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development.

Synthetic biology applied to baculoviruses enables genome optimization through the targeted deletion of nonessential genes, enhancing recombinant protein expression. In this study, the CRISPR/Cas9 system with two tandem sgRNAs was used and validated as an efficient strategy to remove independently large genomic fragments from AcMNPV, all encoding proteins dispensable for budded virus production. The resulting mutant viruses were evaluated for their ability to express eGFP and HRPc in Sf9 cells and in Rachiplusia nu and Spodoptera frugiperda larvae. Deletions of the Ac15–Ac16 and Ac129–Ac131 regions led to significant increases in protein expression in infected cells and in larvae. In contrast, deletion of the Ac136–Ac138 region enhanced expression only in cultured cells but had a negative impact on larvae. Removal of the Ac148–Ac150 fragment caused a marked reduction in expression in both experimental systems. These findings confirm that although some genes are nonessential for systemic infection, their combined deletion can differentially affect recombinant protein expression depending on the host. This study validates not only an effective strategy for developing minimized baculovirus genomes but also the use of dual-guide CRISPR/Cas9 editing as a rapid and precise tool for genome engineering in baculovirus-based expression systems.