Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe₃O₄ (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO₂) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Humanin (HN) is a mitochondrial-derived peptide with robust cytoprotective effects in many cell types. Although the administration of HN analogs has been proposed to treat degenerative diseases, its role in the pathogenesis of cancer is poorly understood. Here, we evaluated whether HN affects the chemosensitivity of glioblastoma (GBM) cells. We found that chemotherapy upregulated HN expression in GBM cell lines and primary cultures derived from GBM biopsies. An HN analog (HNGF6A) boosted chemoresistance, increased the migration of GBM cells and improved their capacity to induce endothelial cell migration and proliferation. Chemotherapy also upregulated FPR2 expression, an HN membrane-bound receptor, and the HNGF6A cytoprotective effects were inhibited by an FPR2 receptor antagonist (WRW4). These effects were observed in glioma cells with heterogeneous genetic backgrounds, i.e., glioma cells with wild-type (wtIDH) and mutated (mIDH) isocitrate dehydrogenase. HN silencing using a baculoviral vector that encodes for a specific shRNA for HN (BV.shHN) reduced chemoresistance, and impaired the migration and proangiogenic capacity of GBM cells. Taken together, our findings suggest that HN boosts the hallmark characteristics of GBM, i.e., chemoresistance, migration and endothelial cell proliferation. Thus, strategies that inhibit the HN/FPR2 pathway may improve the response of GBM to standard therapy

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterial β-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh⁺) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

PURPOSE: The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 (baculoviral IAP repeat-containing protein 6) in breast cancer (BC), focusing particularly on its involvement in the metastatic cascade. METHODS: We analyzed BIRC6 mRNA expression levels and copy number variations in three BC databases from The Cancer Genome Atlas comparing clinical and molecular attributes. Genomic analysis was performed using the cBioPortal platform, whereas transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots, and gene ontology) were performed using USC Xena and R. Statistical significance was set at P < .05. RESULTS: Our bioinformatic analyses showed that there was a differential expression of BIRC6 in cancer samples when compared with normal samples. Copy number variations that involve amplification and gain of BIRC6 gene were correlated with negative hormone receptor tumors, higher prognostic indexes, younger age at diagnosis, and both chemotherapy and radiotherapy administration. Transcriptomic and gene ontology analyses showed that, under conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. CONCLUSION: In summary, our in silico data suggest that BIRC6 plays an antiapoptotic, pro-proliferative, and apparent prometastatic role and could be a relevant molecular target for treatment of BC tumors.